Neisseria meningitidis compositions and methods thereof

ABSTRACT

In one aspect, the invention relates to a composition including a first polypeptide having the sequence set forth in SEQ ID NO: 1 and a second polypeptide having the sequence set forth in SEQ ID NO: 2. In one embodiment, the composition includes about 120 μg/ml of a first polypeptide including the amino acid sequence set forth in SEQ ID NO: 1, 120 μg/ml of a second polypeptide including the amino acid sequence set forth in SEQ ID NO: 2, about 2.8 molar ratio polysorbate-80 to the first polypeptide, about 2.8 molar ratio polysorbate-80 to the second polypeptide, about 0.5 mg/ml aluminum, about 10 mM histidine, and about 150 mM sodium chloride. In one embodiment, a dose of the composition is about 0.5 ml in total volume. In one embodiment, two-doses of the composition induce a bactericidal titer against diverse heterologous subfamily A and subfamily B strains in a human.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a U.S. national phase application under 35 U.S.C. § 371 of International Application No. PCT/IB2016/050829, filed Feb. 16, 2016, which claims the benefit of U.S. Provisional Patent Application 62/118,457, filed on Feb. 19, 2015, and U.S. Provisional Patent Application 62/280,212, filed on Jan. 19, 2016. All of the foregoing applications are hereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to Neisseria meningitidis compositions and methods thereof.

BACKGROUND OF THE INVENTION

Neisseria meningitidis is a Gram-negative encapsulated bacterium that can cause sepsis, meningitis, and death. N. meningitidis can be classified into at least 12 serogroups (including serogroups A, B, C, 29E, H, I, K, L, W-135, X, Y and Z) based on chemically and antigenically distinctive polysaccharide capsules. Strains with five of the serogroups (A, B, C, Y, and W135) are responsible for the majority of disease.

Meningococcal meningitis is a devastating disease that can kill children and young adults within hours despite the availability of antibiotics. There is a need for improved immunogenic compositions against meningococcal serogroups A, B, C, Y, and W135 and/or X.

Currently, a cross-protective vaccine or composition effective against a wide range of MnB isolates is not yet commercially available. For example, published results-to-date relating to a licensed multi-component composition for protection against serogroup B disease has not demonstrated a direct bactericidal immune response against multiple strains expressing heterologous LP2086 (fHBP) variants, at least in adolescents. At most, published results-to-date relating to the multi-component composition for protection against serogroup B disease appear to show immunogenicity against LP2086 (fHBP) variants that are homologous to the LP2086 (fHBP) variant in the multi-component composition. Accordingly, a cross-protective vaccine or composition effective against diverse MnB isolates is needed as is determining real-world vaccine coverage against a panel of diverse or heterologous meningococcal strains (e.g., representing different geographical regions).

SUMMARY OF THE INVENTION

In one aspect, the invention relates to a composition including about 120 μg/ml of a first lipidated polypeptide including the amino acid sequence set forth in SEQ ID NO: 1, 120 μg/ml of a second lipidated polypeptide including the amino acid sequence set forth in SEQ ID NO: 2, about 2.8 molar ratio polysorbate-80 to the first polypeptide, about 2.8 molar ratio polysorbate-80 to the second polypeptide, about 0.5 mg/ml aluminum, about 10 mM histidine, and about 150 mM sodium chloride. In one embodiment, the first dose is about 0.5 ml in total volume. In one embodiment, the composition induces a bactericidal immune response against N. meningitidis serogroup B. In one embodiment, the composition may induce a bactericidal immune response against N. meningitidis serogroup A, C, 29E, H, I, K, L, W-135, X, Y or Z. In one embodiment, the composition does not further include a polypeptide having less than 100% sequence identity to SEQ ID NO: 1. In one embodiment, the composition does not further include a polypeptide having less than 100% sequence identity to SEQ ID NO: 2. In one embodiment, the first polypeptide has a total of 258 amino acids. In one embodiment, the second polypeptide has a total of 261 amino acids. In one embodiment, the composition induces a bactericidal titer of serum immunoglobulin against N. meningitidis serogroup B that is at least 2-fold higher in the human after receiving the first dose than a bactericidal titer of serum immunoglobulin against N. meningitidis serogroup B in the human prior to receiving the first dose, wherein the increase in bactericidal titer is measured under identical conditions in a serum bactericidal assay using human complement. In one embodiment, the first lipidated polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 1. In one embodiment, the second lipidated polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 2.

In another aspect, the invention relates to a method of inducing an immune response against Neisseria meningitidis serogroup B in a human. The method includes administering to the human a first dose and a second dose of an effective amount of a composition, said composition including 120 μg/ml of a first lipidated polypeptide including the amino acid sequence set forth in SEQ ID NO: 1, 120 μg/ml of a second lipidated polypeptide including the amino acid sequence set forth in SEQ ID NO: 2, 2.8 molar ratio polysorbate-80 to the first polypeptide, 2.8 molar ratio polysorbate-80 to the second polypeptide, 0.5 mg/ml aluminum, 10 mM histidine, and 150 mM sodium chloride. In one embodiment, a dose of the composition has a total volume of 0.5 ml. In one embodiment, the human is administered at most two doses of the composition. In one embodiment, the human is not further administered a booster dose of the composition. In one embodiment, the human is administered a third dose of the composition. In one embodiment, the human is not further administered a booster dose of the composition after the third dose. In one embodiment, the human is not further administered a fourth dose of the composition. In one embodiment, the third dose is administered to the human within a period of about 6 months after the first dose. In one embodiment, the second dose is administered at least 30 days after the first dose. In one embodiment, the method further includes administering a third dose of the composition, wherein the third dose is administered at least 90 days after the second dose. In one embodiment, the composition induces a bactericidal titer of serum immunoglobulin against N. meningitidis serogroup B that is at least 2-fold higher in the human after receiving the first dose than a bactericidal titer of serum immunoglobulin against N. meningitidis serogroup B in the human prior to receiving the first dose, when measured under identical conditions in a serum bactericidal assay using human complement. In one embodiment, the immune response is bactericidal against a N. meningitidis serogroup B subfamily A strain that is heterologous to a N. meningitidis strain expressing A05. In one embodiment, the immune response is bactericidal against a N. meningitidis serogroup B subfamily B strain that is heterologous to a N. meningitidis strain expressing B01. In one embodiment, the immune response is bactericidal against a N. meningitidis serogroup B subfamily A strain that is heterologous to N. meningitidis strain M98250771. In one embodiment, the immune response is bactericidal against a N. meningitidis serogroup B subfamily B strain that is heterologous to N. meningitidis strain CDC1127. In a preferred embodiment, the immune response is bactericidal against a N. meningitidis serogroup B subfamily B strain that is heterologous to N. meningitidis strain CDC1573. In one embodiment, the first polypeptide has a total of 258 amino acids. In one embodiment, the second polypeptide has a total of 261 amino acids. In one embodiment, the first lipidated polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 1. In one embodiment, the second lipidated polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 2.

In another aspect, the invention relates to a composition that includes 60 μg of a first lipidated polypeptide including the amino acid sequence set forth in SEQ ID NO: 1, 60 μg of a second lipidated polypeptide including the amino acid sequence set forth in SEQ ID NO: 2, 2.8 molar ratio polysorbate-80 to the first polypeptide, 2.8 molar ratio polysorbate-80 to the second polypeptide, 0.5 mg/ml aluminum, 10 mM histidine, and 150 mM sodium chloride, wherein the composition has a total volume of about 0.5 ml. In one embodiment, the composition induces a bactericidal immune response against a N. meningitidis serogroup B subfamily A strain that is heterologous to a N. meningitidis strain expressing A05. In one embodiment, the composition induces a bactericidal immune response against a N. meningitidis serogroup B subfamily B strain that is heterologous to a N. meningitidis strain expressing B01. In one embodiment, the composition induces a bactericidal titer of serum immunoglobulin against N. meningitidis serogroup B that is at least 2-fold higher in the human after receiving the first dose than a bactericidal titer of serum immunoglobulin against N. meningitidis serogroup B in the human prior to receiving the first dose, when measured under identical conditions in a serum bactericidal assay using human complement. In one embodiment, the composition does not further include a polypeptide having less than 100% sequence identity to SEQ ID NO: 1. In one embodiment, the composition does not further include a polypeptide having less than 100% sequence identity to SEQ ID NO: 2. In one embodiment, the first polypeptide has a total of 258 amino acids. In one embodiment, the second polypeptide has a total of 261 amino acids. In one embodiment, the first lipidated polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 1. In one embodiment, the second lipidated polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 2.

In a further aspect, the invention relates to a method of inducing a bactericidal immune response against a Neisseria meningitidis serogroup B subfamily A strain, and against a Neisseria meningitidis serogroup B subfamily B strain in a human. The method includes administering to the human an effective amount of a N. meningitidis rLP2086 composition, said composition comprising a) a first lipidated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1, and b) a second lipidated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, wherein the method further comprises administering to the human at least one of the following immunogenic compositions within 24 hours of administering said composition against N. meningitidis serogroup B: (i) an immunogenic composition against a Neisseria meningitidis serogroup A strain, a Neisseria meningitidis serogroup C strain, a Neisseria meningitidis serogroup Y strain, and/or a Neisseria meningitidis serogroup W135 strain; (ii) an immunogenic composition against diphtheria, tetanus, and pertussis; (iii) an immunogenic composition against hepatitis A virus; (iv) an immunogenic composition against human papillomavirus; (v) an immunogenic composition against diphtheria, tetanus, pertussis and poliomyelitis; or (vi) any combination thereof. “Immunogenic” refers to an ability to induce an immune response.

In one aspect, the invention relates to a method of inducing a bactericidal immune response against a N. meningitidis serogroup B subfamily A strain, and against a N. meningitidis serogroup B subfamily B strain in a human. The method includes administering to the human an effective amount of a N. meningitidis rLP2086 composition, said composition including a) a first lipidated polypeptide including the amino acid sequence set forth in SEQ ID NO: 1, and b) a second lipidated polypeptide including the amino acid sequence set forth in SEQ ID NO: 2, wherein the method further includes concomitantly administering to the human at least one of the following additional immunogenic compositions: (i) an immunogenic composition against a N. meningitidis serogroup A strain, a N. meningitidis serogroup C strain, a N. meningitidis serogroup Y strain, and/or a N. meningitidis serogroup W135 strain; (ii) an immunogenic composition against diphtheria, tetanus, and pertussis; (iii) an immunogenic composition against hepatitis A virus; (iv) an immunogenic composition against human papillomavirus; (v) an immunogenic composition against diphtheria, tetanus, pertussis and poliomyelitis; or (vi) any combination thereof.

In another aspect, the invention relates to use of an effective amount of a N. meningitidis rLP2086 composition and at least one additional immunogenic composition concomitantly for inducing a bactericidal immune response against a Neisseria meningitidis serogroup B subfamily A strain, and against a Neisseria meningitidis serogroup B subfamily B strain in a human, wherein said N. meningitidis rLP2086 composition comprises a) a first lipidated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1, and b) a second lipidated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, wherein the at least one additional immunogenic composition is: (i) an immunogenic composition against a Neisseria meningitidis serogroup A strain, a Neisseria meningitidis serogroup C strain, a Neisseria meningitidis serogroup Y strain, and/or a Neisseria meningitidis serogroup W135 strain; (ii) an immunogenic composition against diphtheria, tetanus, and pertussis; (iii) an immunogenic composition against hepatitis A virus; (iv) an immunogenic composition against human papillomavirus; (v) an immunogenic composition against diphtheria, tetanus, pertussis and poliomyelitis; or (vi) any combination thereof.

In one embodiment, the N. meningitidis rLP2086 composition further includes polysorbate-80. In one embodiment, the N. meningitidis rLP2086 composition further includes aluminum. In one embodiment, the N. meningitidis rLP2086 composition further includes histidine buffer. In one embodiment, the N. meningitidis rLP2086 composition further includes sodium chloride. In one embodiment, the N. meningitidis rLP2086 composition includes about 120 μg/ml of the first polypeptide; about 120 μg/ml of the second polypeptide; about 2.8 molar ratio of polysorbate-80; about 0.5 mg/ml aluminum; about 10 mM histidine; and about 150 mM sodium chloride. In one embodiment, the N. meningitidis rLP2086 composition includes about 60 μg of the first polypeptide; about 60 μg of the second polypeptide; about 18 μg polysorbate-80; about 250 μg aluminum; about 780 μg histidine; and about 4380 μg sodium chloride.

In one embodiment, the N. meningitidis rLP2086 composition induces a bactericidal immune response against at least one of N. meningitidis serogroup B A22, A56, B24, B44 strains, or any combination thereof. In one embodiment, the N. meningitidis rLP2086 composition induces a bactericidal immune response against N. meningitidis serogroup B A22. In one embodiment, the N. meningitidis rLP2086 composition induces a bactericidal immune response against N. meningitidis serogroup B B24. In one embodiment, the N. meningitidis rLP2086 composition induces a bactericidal immune response against at least one of N. meningitidis serogroup B B24, B16, B44, A22, B03, B09, A12, A19, A05, A07, B153 strains, or any combination thereof.

In one embodiment, the method further includes inducing an immune response against at least one of a Neisseria meningitidis serogroup A strain, a Neisseria meningitidis serogroup C strain, a Neisseria meningitidis serogroup Y strain, and/or a Neisseria meningitidis serogroup W135 strain, or any combination thereof.

In one embodiment, the method includes administering to the human an additional immunogenic composition including a mixture of four distinct and separately made protein-capsular polysaccharide conjugates, wherein the first conjugate includes purified N. meningitidis capsular polysaccharide of serogroup W-135 conjugated to a carrier protein, the second conjugate includes purified N. meningitidis capsular polysaccharide of serogroup Y conjugated to a carrier protein, the third conjugate includes purified N. meningitidis capsular polysaccharide of serogroup A conjugated to a purified carrier protein, and the fourth conjugate includes purified N. meningitidis capsular polysaccharide of serogroup C conjugated to a carrier protein, wherein the carrier protein is selected from the group consisting of diphtheria toxoid, CRM197, and tetanus toxoid. In one embodiment, the carrier protein is diphtheria toxoid.

In one embodiment, the immunogenic composition is a liquid composition.

In one embodiment, the immunogenic composition is not lyophilized.

In one embodiment, the method further includes inducing an immune response against at least one of human papillomavirus type 6, 11, 16, 18, or any combination thereof. In one embodiment, the method further includes inducing an immune response against at least one of diphtheria, tetanus, pertussis, poliomyelitis, or any combination thereof in the human.

In one embodiment, the effective amount of the N. meningitidis rLP2086 composition includes one dose. In one embodiment, the effective amount of the N. meningitidis rLP2086 composition includes two doses. In one embodiment, the effective amount of the N. meningitidis rLP2086 composition further includes a booster dose. In one embodiment, the effective amount of the N. meningitidis rLP2086 composition includes at most three doses.

In one embodiment, the N. meningitidis rLP2086 composition is administered within 24 hours of administration of the additional composition. In one embodiment, the N. meningitidis rLP2086 composition is simultaneously administered to the additional composition. In one embodiment, a first dose of the N. meningitidis rLP2086 composition is concomitantly administered to the additional composition. In one embodiment, the first dose of the N. meningitidis rLP2086 composition is simultaneously administered to the additional composition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1—Proportion of Subjects Achieving hSBA Titers ≥LLOQ. hSBA=serum bactericidal assay using human complement; LLOQ=lower limit of quantitation.

FIG. 2—Percentage of subjects achieving 4× rise in hSBA titers to Princeton University Outbreak Strains and UCSB Outbreak Strains of Individual Human Subjects Following Immunization With rLP2086 (Study B1971012—described in Example 5, Example 6). Serum samples from nine human subjects immunized with bivalent rLP2086 in clinical study B1971012 were evaluated in exploratory hSBAs using MnB outbreak strains from Princeton University and from UCSB. See Example 9.

SEQUENCE IDENTIFIERS

SEQ ID NO: 1 sets forth the amino acid sequence for a recombinant N. meningitidis, serogroup B, 2086 variant A05 polypeptide antigen.

SEQ ID NO: 2 sets forth the amino acid sequence for a recombinant N. meningitidis, serogroup B, 2086 variant B01 polypeptide antigen.

SEQ ID NO: 3 sets forth the amino acid residues at positions 1-4 of SEQ ID NO: 1 and SEQ ID NO: 2.

SEQ ID NO: 4 sets forth the amino acid sequence of the N-terminus of a recombinant Neisserial Subfamily A LP2086 polypeptide (rLP2086) (A05) polypeptide antigen.

SEQ ID NO: 5 sets forth the amino acid sequence of the N-terminus of Neisserial Subfamily A LP2086 M98250771 polypeptide (A05) polypeptide antigen.

SEQ ID NO: 6 sets forth the the amino acid sequence for N. meningitidis, serogroup B, 2086 variant B153.

SEQ ID NO: 7 sets forth the amino acid sequence for N. meningitidis, serogroup B, 2086 variant A04.

SEQ ID NO: 8 sets forth the amino acid sequence for N. meningitidis, serogroup B, 2086 variant A05

SEQ ID NO: 9 sets forth the amino acid sequence for N. meningitidis, serogroup B, 2086 variant A12.

SEQ ID NO: 10 sets forth the amino acid sequence for N. meningitidis, serogroup B, 2086 variant A22.

SEQ ID NO: 11 sets forth the amino acid sequence for N. meningitidis, serogroup B, 2086 variant B02.

SEQ ID NO: 12 sets forth the amino acid sequence for N. meningitidis, serogroup B, 2086 variant B03.

SEQ ID NO: 13 sets forth the amino acid sequence for N. meningitidis, serogroup B, 2086 variant B09.

SEQ ID NO: 14 sets forth the amino acid sequence for N. meningitidis, serogroup B, 2086 variant B22.

SEQ ID NO: 15 sets forth the amino acid sequence for N. meningitidis, serogroup B, 2086 variant B24.

SEQ ID NO: 16 sets forth the amino acid sequence for N. meningitidis, serogroup B, 2086 variant B44.

SEQ ID NO: 17 sets forth the amino acid sequence for N. meningitidis, serogroup B, 2086 variant B16.

SEQ ID NO: 18 sets forth the amino acid sequence for N. meningitidis, serogroup B, 2086 variant A07.

SEQ ID NO: 19 sets forth the amino acid sequence for N. meningitidis, serogroup B, 2086 variant A19.

SEQ ID NO: 20 sets forth the amino acid sequence for N. meningitidis, serogroup B, 2086 variant A06.

SEQ ID NO: 21 sets forth the amino acid sequence for N. meningitidis, serogroup B, 2086 variant A15.

SEQ ID NO: 22 sets forth the amino acid sequence for N. meningitidis, serogroup B, 2086 variant A29.

SEQ ID NO: 23 sets forth the amino acid sequence for N. meningitidis, serogroup B, 2086 variant B15.

DETAILED DESCRIPTION OF THE INVENTION

The inventors surprisingly discovered an rLP2086 composition that includes a first lipidated polypeptide including the amino acid sequence set forth in SEQ ID NO: 1 and a second lipidated polypeptide including the amino acid sequence set forth in SEQ ID NO: 2. The composition has an acceptable safety profile in humans and the composition surprisingly elicits a broadly cross-reactive bactericidal immune response in humans against at least more than two diverse Neisseria meningitidis strains.

The inventors also discovered that robust immune responses against N. meningitidis serogroup B were generated even after concomitant administration of the rLP2086 composition and at least one of the following immunogenic compositions: (i) an immunogenic composition against a N. meningitidis serogroup A strain, a N. meningitidis serogroup C strain, a N. meningitidis serogroup Y strain, and/or a N. meningitidis serogroup W135 strain; (ii) an immunogenic composition against diphtheria, tetanus, and pertussis; (iii) an immunogenic composition against hepatitis A virus; (iv) an immunogenic composition against human papillomavirus; (v) an immunogenic composition against diphtheria, tetanus, pertussis and poliomyelitis; or (vi) any combination thereof.

The concomitant administration of at least the rLP2086 composition and a meningococcal Groups A, C, Y and W-135 polysaccharide conjugate vaccine (MCV4) also surprisingly generated an immune response at least against meningococcal Groups A, C, Y and W-135 in addition to meningococcal Group B. The immune responses against the meningococcal Groups A, C, Y and W-135 after concomitant administration of the rLP2086 composition and the MCV4 composition were noninferior when compared to the immune response generated by an administration of the MCV4 composition in the absence of the rLP2086 composition.

The concomitant administration of at least the rLP2086 composition, a meningococcal Groups A, C, Y and W-135 polysaccharide conjugate vaccine (MCV4), and a tetanus, diphtheria, and acellular pertussis (Tdap) vaccine also surprisingly generated an immune response at least against tetanus, diphtheria, and acellular pertussis in addition to meningococcal Groups A, C, Y and W-135 and to meningococcal Group B. The immune responses against the Tdap and meningococcal Groups A, C, Y and W-135 after concomitant administration of the rLP2086 composition, the MCV4 composition, and Tdap composition were noninferior when compared to the immune response generated by an administration of the MCV4 composition and Tdap composition in the absence of the rLP2086 composition.

Moreover, the inventors discovered that the rLP2086 composition can be safely administered to humans concomitantly with an immunogenic composition against hepatitis A virus.

The inventors further discovered that a 2-dose administration schedule and a 3-dose administration schedule surprisingly yielded hSBA titers of against test strains from N. meningitidis serogroup B, with vaccine heterologous LP2086 (factor H binding protein (fHBP)) subfamilies A and B in a high proportion of human subjects. A 3-dose administration schedule may provide broader protection in humans against diverse MnB clinical strains, when compared to a 2-dose administration schedule.

The inventors also surprisingly discovered that robust immune responses against human papillomavirus and N. meningitidis serogroup B were generated after concomitant administration of the rLP2086 composition and a quadrivalent

immunogenic composition against human papillomavirus (HPV4). For example, a concomitant administration of the rLP2086 composition and HPV4 composition generated an immune response at least against N. meningitidis serogroup B test strains expressing fHBPs that are heterologous to those fHBPs in the rLP2086 composition. Such heterologous test strains include wild-type N. meningitidis serogroup B strains that express A22 fHBP, A56 fHBP, B24 fHBP, or B44 fHBP, which are each heterologous to the fHBPs in the rLP2086 composition. See WO/2012/032489, WO/2013/132452, US patent publication number US20120093852, and US patent publication number US20130243807, which describe variant fHBP proteins, including A22 fHBP, A56 fHBP, B24 fHBP, and B44 fHBP, among others. These references are each incorporated by reference in their entirety. The concomitant administration also surprisingly generated an immune response at least against HPV types 6, 11, 16, and/or 18. The immune responses against the HPV types after concomitant administration of the rLP2086 composition and the HPV4 composition were noninferior when compared to the immune response generated by an administration of the HPV4 composition in the absence of the rLP2086 composition.

In addition, the inventors surprisingly discovered that robust immune responses against diphtheria, tetanus, pertussis and poliomyelitis and N. meningitidis serogroup B were generated after concomitant administration of the rLP2086 composition and an immunogenic composition against diphtheria, tetanus, pertussis and poliomyelitis. For example, a concomitant administration of the rLP2086 composition and REPEVAX composition generated an immune response at least against N. meningitidis serogroup B test strains expressing fHBPs that are heterologous to those fHBPs in the rLP2086 composition. The concomitant administration also surprisingly generated an immune response at least against the 9 antigens in REPEVAX: diphtheria, tetanus, pertussis toxoid, pertussis filamentous hemagglutinin, pertussis pertactin, pertussis fimbrial agglutinogens type 2+3, poliovirus type 1, poliovirus type 2, poliovirus type 3. The immune responses against the REPEVAX antigens after concomitant administration of the rLP2086 composition and the REPEVAX composition were noninferior when compared to the immune response generated by an administration of the REPEVAX composition in the absence of the rLP2086 composition.

Composition and Vaccine

In one aspect, the invention relates to a composition against Neisseria meningitidis. The composition includes a first lipidated polypeptide having the amino acid sequence set forth in SEQ ID NO: 1, and a second lipidated polypeptide having the amino acid sequence set forth in SEQ ID NO: 2.

The inventors surprisingly discovered a single N. meningitidis polypeptide component that induces an effective broadly protective immune response against multiple strains of N. meningitidis serogroup B. Accordingly, in one embodiment, the composition does not include a fusion protein. In one embodiment, the composition does not include a chimeric protein. In one embodiment, the composition does not include a hybrid protein. In one embodiment, the composition does not further include a peptide fragment. In another embodiment, the composition does not further include a Neisserial polypeptide that is not fHBP. For example, in one embodiment, the composition does not include a PorA protein. In another embodiment, the composition does not include a NadA protein. In another embodiment, the composition does not further include a Neisserial heparin binding antigen (NHBA). In another embodiment, the composition does not further include a Neisserial outer membrane vesicle (OMV). In a preferred embodiment, the composition does not further include antigens, other than the first polypeptide and the second polypeptide.

In another aspect, the inventors surprisingly discovered that polypeptide antigens derived from at most two N. meningitidis serogroup B strains induces an immune response against multiple strains of N. meningitidis serogroup B. Accordingly, in one embodiment, the composition does not further include a polypeptide that is not derived from N. meningitidis serogroup B subfamily A M98250771 strain and/or N. meningitidis serogroup B subfamily B CDC1573 strain.

In one embodiment, the composition does not further include a polypeptide having less than 100% sequence identity to SEQ ID NO: 1. In another embodiment, the composition does not further include a polypeptide having less than 100% sequence identity to SEQ ID NO: 2. For example, the composition does not further include a polypeptide having less than 100% sequence identity to the full length of SEQ ID NO: 1 and/or SEQ ID NO: 2.

In one embodiment, the composition further includes polysorbate-80, aluminum, histidine, and sodium chloride. In one embodiment, the composition includes about 60 μg of a first lipidated polypeptide including the amino acid sequence set forth in SEQ ID NO: 1, about 60 μg of a second lipidated polypeptide including the amino acid sequence set forth in SEQ ID NO: 2, 2.8 molar ratio of polysorbate-80 to each polypeptide, 0.5 mg aluminum/ml as aluminum phosphate, 10 mM histidine, and 150 mM sodium chloride, wherein the composition preferably has a total volume of about 0.5 ml.

In another aspect, the composition includes about 120 μg/ml of a first lipidated polypeptide including the amino acid sequence set forth in SEQ ID NO: 1, about 120 μg/ml of a second lipidated polypeptide including the amino acid sequence set forth in SEQ ID NO: 2, 2.8 molar ratio of polysorbate-80 to each polypeptide, 0.5 mg aluminum/ml as aluminum phosphate, 10 mM histidine, and 150 mM sodium chloride.

In a further aspect, the composition includes a) 60 μg of a first lipidated polypeptide including the amino acid sequence set forth in SEQ ID NO: 1; b) 60 μg of a second lipidated polypeptide including the amino acid sequence set forth in SEQ ID NO: 2; c) 18 μg polysorbate-80; d) 250 μg aluminum; e) 780 μg histidine, and; f) 4380 μg sodium chloride.

In an exemplary embodiment, the composition includes about 60 μg of a first lipidated polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1, about 60 μg of a second lipidated polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2, 2.8 molar ratio of polysorbate-80 to first lipidated polypeptide and to second lipidated polypeptide, 0.5 mg/ml aluminum phosphate, 10 mM histidine, and 150 mM sodium chloride, wherein the composition has a total volume of about 0.5 ml. In the exemplary embodiment, the composition is a sterile isotonic buffered liquid suspension. In the exemplary embodiment, the composition has a pH 6.0. In the exemplary embodiment, the first polypeptide and the second polypeptide are adsorbed to aluminum.

In one embodiment, the composition has a total volume of about 0.5 ml. In one embodiment, a first dose of the composition has a total volume of about 0.5 ml. A “first dose” refers to the dose of the composition that is administered on Day 0. A “second dose” or “third dose” refers to the dose of the composition that is administered subsequently to the first dose, which may or may not be the same amount as the first dose.

The composition is immunogenic after administration of a first dose to a human. In one embodiment, the first dose is about 0.5 ml in total volume.

The composition induces a bactericidal titer of serum immunoglobulin against N. meningitidis serogroup B that is at least greater than 1-fold higher, preferably at least 2-fold higher, in the human after receiving the first dose than a bactericidal titer of serum immunoglobulin against N. meningitidis serogroup B in the human prior to receiving the first dose, when measured under identical conditions in a serum bactericidal assay using human complement (hSBA).

In a preferred embodiment, the bactericidal titer or bactericidal immune response is against a N. meningitidis serogroup B subfamily A strain and against a N. meningitidis serogroup B subfamily B strain. Most preferably, the bactericidal titer or bactericidal immune response is at least against N. meningitidis serogroup B, subfamily B, B01 strain.

In another preferred embodiment, the bactericidal titer or bactericidal immune response is at least against N. meningitidis serogroup B, subfamily B, B24 strain. In another preferred embodiment, the bactericidal titer or bactericidal immune response is at least against N. meningitidis serogroup B, subfamily A, A22 strain.

In one embodiment, the composition induces a bactericidal titer of serum immunoglobulin against N. meningitidis serogroup B that is at least greater than 1-fold, such as, for example, at least 1.01-fold, 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, or 16-fold higher in the human after receiving a dose of the composition than a bactericidal titer of serum immunoglobulin against N. meningitidis serogroup B in the human prior to receiving said dose, when measured under identical conditions in a serum bactericidal assay using human complement.

In one embodiment, the composition is an immunogenic composition for a human. In another embodiment, the composition is a vaccine. A “vaccine” refers to a composition that includes an antigen, which contains at least one epitope that induces an immune response that is specific for that antigen. The vaccine may be administered directly into the subject by subcutaneous, oral, oronasal, or intranasal routes of administration. Preferably, the vaccine is administered intramuscularly. In one embodiment, the composition is a vaccine for humans. In one embodiment, the composition is an immunogenic composition against N. meningitidis.

In one embodiment, the composition is a liquid composition. In a preferred embodiment, the composition is a liquid suspension composition. In another preferred embodiment, the composition is not lyophilized.

First Polypeptide

In one embodiment, the composition includes a first polypeptide having the amino acid sequence set forth in SEQ ID NO: 1. In one preferred embodiment, the composition includes about 60 μg of a first polypeptide including the amino acid sequence set forth in SEQ ID NO: 1, wherein the composition preferably has a total volume of 0.5 ml. In another embodiment, the composition includes about 120 μg/ml of a first polypeptide including the amino acid sequence set forth in SEQ ID NO: 1. The polypeptide is a modified factor H binding protein (fHBP) from N. meningitidis strain M98250771. A description of fHBP is disclosed in WO2012032489 and US patent publication US 2012/0093852, which are each incorporated by reference in their entirety. The polypeptide is N-terminally lipidated with three predominant fatty acids C16:0, C16:1, and C18:1 covalently linked at three positions of the polypeptide. The first polypeptide includes a total of 258 amino acids.

The first polypeptide includes two modifications introduced in the N-terminal region of the polypeptide, as compared to the corresponding wild-type sequence from N. meningitidis strain M98250771. A glycine in the second position is added as a consequence of introducing a cloning site. A second modification includes the deletion of four amino acids. Accordingly, in one embodiment, the first polypeptide includes a C-G-S-S sequence (SEQ ID NO: 3) at the N-terminus. See SEQ ID NO: 1, first four amino acid residues.

The N-terminal differences between the first polypeptide sequence and the wild-type Neisserial sequence is shown below. Accordingly, in one embodiment, the first polypeptide includes at least the first 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or more amino acid residues of the amino acid sequence set forth in SEQ ID NO: 1. Preferably, the first polypeptide includes at least the first 4, more preferably at least the first 6, and most preferably, at least the first 8 amino acid residues of SEQ ID NO: 1.

Comparison of Predicted N-Terminal Sequences of Recombinant and Neisserial

Subfamily A LP2086 Polypeptide

rLP2086 M98250771 (SEQ ID NO: 4) CGSS-----GGGGVAAD Neisserial LP2086 M98250771 (SEQ ID NO: 5) C-SSGS-GSGGGGVAAD >A05  (SEQ ID NO: 1) CGSSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLS AQGAEKTFKVGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEF QIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPS GKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELASAE LKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIREKVH EIGIAGKQ

In one embodiment, the first polypeptide includes the amino acid sequence set forth in SEQ ID NO: 1. In one embodiment, the first polypeptide has a total of 258 amino acids. In one embodiment, the first polypeptide does not include an amino acid sequence having less than 100% sequence identity to SEQ ID NO: 1. In another embodiment, the first polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 1. In another embodiment, the first polypeptide includes the amino acid sequence KDN. See for example, amino acid residues 73-75 of SEQ ID NO: 1. In another embodiment, the first polypeptide includes the amino acid sequence set forth in SEQ ID NO: 3 at the N-terminus of the polypeptide. In another embodiment, the first polypeptide includes the amino acid sequence set forth in SEQ ID NO: 4 at the N-terminus of the polypeptide.

In a preferred embodiment, the first polypeptide is readily expressed in a recombinant host cell using standard techniques known in the art. In another preferred embodiment, the first polypeptide includes a bactericidal epitope on the N- and/or C-domain of SEQ ID NO: 1. In one embodiment, the first polypeptide includes at least the first 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acid residues of the amino acid sequence set forth in SEQ ID NO: 1. Preferably, the first polypeptide includes at least the first 2, more preferably at least the first 4, and most preferably, at least the first 8 amino acid residues of SEQ ID NO: 1.

In another embodiment, the first polypeptide includes at least the last 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acid residues of the amino acid sequence set forth in SEQ ID NO: 1.

Second Polypeptide

In one embodiment, the composition includes a second polypeptide having the amino acid sequence set forth in SEQ ID NO: 2. In one preferred embodiment, the composition includes about 60 μg of a second polypeptide including the amino acid sequence set forth in SEQ ID NO: 2, wherein the composition preferably has a total volume of 0.5 ml. In another embodiment, the composition includes 120 μg/ml of a second polypeptide including the amino acid sequence set forth in SEQ ID NO: 2. The polypeptide is a factor H binding protein (fHBP) from N. meningitidis strain CDC1573. A description of fHBP is disclosed in WO2012032489 and US patent publication US 2012/0093852, which are each incorporated by reference in their entirety. The polypeptide is N-terminally lipidated with three predominant fatty acids C16:0, C16:1, and C18:1 covalently linked at three positions of the polypeptide. The second polypeptide includes a total of 261 amino acids. In one embodiment, the second polypeptide includes a C-G-S-S sequence (SEQ ID NO: 3) at the N-terminus. See the first four amino acid residues of SEQ ID NO: 2.

>B01  (SEQ ID NO: 2) CGSSGGGGSGGGGVTADIGTGLADALTAPLDHKDKGLKSLTLEDSISQNG TLTLSAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESG EFQVYKQSHSALTALQTEQEQDPEHSEKMVAKRRFRIGDIAGEHTSFDKL PKDVMATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDLA VAYIKPDEKHHAVISGSVLYNQDEKGSYSLGIFGEKAQEVAGSAEVETAN GIHHIGLAAKQ

In one embodiment, the second polypeptide includes the amino acid sequence set forth in SEQ ID NO: 2. In one embodiment, the second polypeptide has a total of 261 amino acids. In one embodiment, the second polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 2. In another embodiment, the second polypeptide does not further include a polypeptide having less than 100% sequence identity to SEQ ID NO: 2. In a preferred embodiment, the first polypeptide and the second polypeptide includes a C-G-S-S (SEQ ID NO: 3) sequence at the N-terminus of the respective polypeptide.

In a preferred embodiment, the second polypeptide is readily expressed in a recombinant host cell using standard techniques known in the art. In another preferred embodiment, the second polypeptide includes a bactericidal epitope on the N- and/or C-domain of SEQ ID NO: 2. In one embodiment, the second polypeptide includes at least the first 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acid residues of the amino acid sequence set forth in SEQ ID NO: 2. Preferably, the second polypeptide includes at least the first 2, more preferably at least the first 4, and most preferably, at least the first 8 amino acid residues of SEQ ID NO: 2.

In another embodiment, the first polypeptide includes at least the last 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acid residues of the amino acid sequence set forth in SEQ ID NO: 2.

Polysorbate-80

Polysorbate 80 (PS-80) is a non-ionic surfactant. Accelerated stability studies using an in vitro monoclonal antibody based potency assay demonstrated instability of the subfamily B protein at higher molar ratios of PS-80 to MnB rLP2086 protein in the final formulation. Further experiments with varying ratios of PS-80 have demonstrated that the optimal molar ratio of PS-80 to MnB rLP2086 protein is approximately 2.8±1.4 to retain potency.

The concentration of PS-80 in the composition is dependent on a molar ratio of PS-80 to the polypeptide. In one embodiment, the composition includes a 2.8±1.4 molar ratio of PS-80 to the first polypeptide and to the second polypeptide. In one embodiment, the composition includes a 2.8±1.1 molar ratio of PS-80 to the first polypeptide and to the second polypeptide. In one embodiment, the composition includes at least 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, or 3.3 molar ratio of PS-80 to polypeptide. Preferably, the composition includes a 2.8 molar ratio of PS-80 to polypeptide.

The PS-80 to polypeptide molar ratio is determined by calculation from the measured concentration of PS-80 and the measured total polypeptide concentration, in which both values are expressed in moles. For example, PS-80 to Protein molar ratio is determined by calculation of the measured concentration of PS-80 (e.g., by reverse phase high pressure liquid chromatography (RP-HPLC)) to the measured total protein concentration (e.g., by ion exchange-high pressure liquid chromatography (IEX-HPLC)) in the final drug substance, where both values are expressed in moles.

A RP-HPLC is used to quantitate the concentration of Polysorbate 80 in vaccine formulations. The concentration of detergent is determined by saponification of the fatty acid moiety; Polysorbate 80 is converted to free oleic acid by alkaline hydrolysis at 40° C. The sample is separated by RP-HPLC using a C18 column and quantitated using a UV detector at a wavelength of 200 nm.

The first and the second polypeptides are resolved by anion-exchange HPLC. rLP2086(fHBP) Subfamily A and B proteins elute at distinct retention times and are quantitated using a standard curve generated against the respective rLP2086 protein reference material.

The term “molar ratio” and a description of an immunogenic composition including a fHBP and PS-80 is further disclosed in WO2012025873 and US patent publication US 2013/0171194, which are each incorporated by reference in their entirety.

The term “molar ratio” as used herein refers to the ratio of the number of moles of two different elements in a composition. In some embodiments, the molar ratio is the ratio of moles of detergent to moles of polypeptide. In some embodiments, the molar ratio is the ratio of moles of PS-80 to moles of protein. In one embodiment, based on the protein and Polysorbate 80 concentrations, the Molar Ratio may be calculated using the following equation:

${{Molar}\mspace{14mu}{Ratio}} = {\frac{\%\mspace{14mu}{PS}\text{-}80}{{mg}\text{/}{ml}\mspace{14mu}{protein}} \times 216}$

In one embodiment, the composition includes about 0.0015, 0.0017, 0.0019, 0.0021, 0.0023, 0.0025, 0.0027, 0.0029, 0.0031, 0.0033, 0.0035, 0.0037, 0.0039, 0.0041, 0.0043, 0.0045, 0.0047, 0.0049, 0.0051 mg/mL PS-80. Preferably, the composition includes about 0.0035 mg/mL PS-80.

In another embodiment, the composition includes about 10 μg, 11 μg, 12 μg, 13 μg, 14 μg, 15 μg, 16 μg, 17 μg, 18 μg, 19 μg, 20 μg, 21 μg, 22 μg, 23 μg, 24 μg, or 25 μg PS-80. In a preferred embodiment, the composition includes about 18 μg PS-80.

In another embodiment, the composition includes a PS-80 concentration ranging from 0.0005% to 1%. For example, the PS-80 concentration in the composition may be at least 0.0005%, 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, or 1.1% PS-80. In a preferred embodiment, the composition includes about 0.07% PS-80.

Any Minimum Value May be Combined with any Maximum Value Described Herein to Define a Range.Aluminum

The composition preferably includes about 0.5 mg/ml aluminum phosphate. In one embodiment, the composition includes about 0.5 mg aluminum/ml as aluminum phosphate. AlPO₄ at 0.50 mg/ml is added as a stabilizer to provide enhanced manufacturability and stability. This concentration maintains binding (90% binding or better) of the subfamily A and B proteins to aluminum.

The process for producing an aluminum phosphate is described in US patent publication US 2009/0016946, which is incorporated by reference in its entirety.

In one embodiment, the composition does not further include a multivalent cation, other than aluminum. In one embodiment, the composition does not further include Al(OH)₃ or Al(SO₄)₃.

Excipients

In one embodiment, the composition includes histidine. In one embodiment, the composition includes sodium chloride. The composition preferably includes about 10 mM histidine, and about 150 mM sodium chloride. In one embodiment, the composition includes 10 mM histidine and 150 mM sodium chloride.

In another embodiment, the composition includes about 650 μg, 660 μg, 670 μg, 680 μg, 690 μg, 700 μg, 710 μg, 720 μg, 730 μg, 740 μg, 750 μg, 760 μg, 770 μg, 780 μg, 790 μg, 800 μg, 810 μg, 820 μg, 830 μg, 840 μg, or 850 μg of histidine. Preferably, the composition includes about 780 μg histidine. Any minimum value may be combined with any maximum value described herein to define a range.

In one embodiment, the composition includes a tris, phosphate, or succinate buffer. In a preferred embodiment, the composition does not include tris buffer. In a preferred, the composition does not include phosphate buffer. In one preferred embodiment, the composition does not include succinate buffer. In a preferred embodiment, the composition includes histidine buffer.

In a preferred embodiment, the pH of the composition is between 6.0 and 7.0, most preferably pH 6.0. In one embodiment, the pH of the composition is at most 6.1.

Bactericidal Activity

Immune response induced by administering the composition to a human is determined using a serum bactericidal assay using human complement (hSBA) against four N. meningitidis serogroup B (MnB) strains. The 4 MnB strains used in the hSBA were selected from a strain pool. The strain pool represented a collection of systematically collected clinically relevant N. meningitidis serogroup B strains from the US and Europe. Two of the 4 strains for the SBA are from N. meningitidis serogroup B LP2086 (fHBP) subfamily A, and another two of the 4 strains are from N. meningitidis serogroup B LP2086(fHBP) subfamily B.

The high proportion of hSBA response to all test strains, especially strains expressing lipoprotein 2086 variants with sequences heterologous to the first polypeptide suggests that the composition is a broadly protective vaccine and that two doses are sufficient to confer high seroprotection at least against N. meningitidis serogroup B subfamily A strains.

The high proportion of hSBA response to all test strains, especially strains expressing lipoprotein 2086 variants with sequences heterologous to both the first polypeptide and the second polypeptide suggests that the composition is a broadly protective vaccine and that at most three doses within about a 6 month period are sufficient to confer high seroprotection against N. meningitidis serogroup B strains expressing rLP2086 (FHBP) subfamily A and/or subfamily B.

In one embodiment, the hSBA strain is an LP2086 (fHBP) subfamily A strain. In one embodiment, the hSBA strain is an LP2086 (fHBP) subfamily A strain that expresses a lipoprotein 2086 variant that is heterologous to a N. meningitidis strain expressing A05. For example, in one embodiment, the hSBA strain is an LP2086 (fHBP) subfamily A strain that expresses a lipoprotein 2086 variant that is heterologous to strain M98250771. In one embodiment, the hSBA strain is an LP2086 (fHBP) A22 strain. In another embodiment, the hSBA strain is an LP2086 (fHBP) A56 strain. In a further embodiment, the hSBA strains are LP2086 (fHBP) A22 and LP2086 (fHBP) A56 strains. In another embodiment, the hSBA strain is an LP2086 A04 strain. In one embodiment, the hSBA strain is an LP2086 A05 strain. In one embodiment, the hSBA strain is an LP2086 A12 strain. In one embodiment, the hSBA strain is an LP2086 A22 strain. In one embodiment, the hSBA strain is an LP2086 A12 strain. In one embodiment, the hSBA strain is an LP2086 A04 strain. In one embodiment, the hSBA strain is an LP2086 A19 strain. In one embodiment, the hSBA strain is an LP2086 A07 strain. In a further embodiment, the hSBA strains include A22, A12, A19, A05, and A07, or any combination thereof. In one embodiment, the hSBA strains include A06, A15, and A29, or any combination thereof.

In one embodiment, the immune response is bactericidal against a N. meningitidis serogroup B subfamily A strain that is heterologous to a N. meningitidis strain expressing A05. In one embodiment, the immune response is against N. meningitidis serogroup B A22 strain. In one embodiment, the immune response is against N. meningitidis serogroup B A56 strain. In one embodiment, the immune response is against N. meningitidis serogroup B A06 strain. In one embodiment, the immune response is against N. meningitidis serogroup B A15 strain. In one embodiment, the immune response is against N. meningitidis serogroup B A29 strain. In one embodiment, the immune response is against N. meningitidis serogroup B A62 strain. In one embodiment, the immune response is bactericidal against a N. meningitidis serogroup B subfamily A strain that is heterologous to N. meningitidis strain M98250771. In one embodiment, the immune response is bactericidal against a N. meningitidis serogroup B subfamily A strain that expresses a factor H binding protein including an amino acid sequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the first polypeptide. In another embodiment, the immune response is bactericidal against a N. meningitidis serogroup B subfamily A strain that expresses a factor H binding protein including an amino acid sequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a factor H binding protein expressed by N. meningitidis strain M98250771. In a preferred embodiment, the immune response is bactericidal against a N. meningitidis serogroup B subfamily A strain that expresses a factor H binding protein including an amino acid sequence that has at least 80%, more preferably at least 84%, identity to a factor H binding protein expressed by N. meningitidis strain M98250771.

In another embodiment, the immune response is bactericidal against a N. meningitidis serogroup B subfamily A strain that expresses a factor H binding protein including an amino acid sequence that has at most 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the first polypeptide. In another embodiment, the immune response is bactericidal against a N. meningitidis serogroup B subfamily A strain that expresses a factor H binding protein including an amino acid sequence that has at most 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a factor H binding protein expressed by N. meningitidis strain M98250771. In a preferred embodiment, the immune response is bactericidal against a N. meningitidis serogroup B subfamily A strain that expresses a factor H binding protein including an amino acid sequence that has at most 99%, more preferably at most 85%, identity to a factor H binding protein expressed by N. meningitidis strain M98250771. Any minimum value may be combined with any maximum value described herein to define a range.

In one embodiment, the hSBA strain is an LP2086 (fHBP) subfamily B strain. In one embodiment, the hSBA strain is an LP2086 (fHBP) subfamily B strain that expresses a lipoprotein 2086 variant that is heterologous to a N. meningitidis strain expressing B01. For example, in one embodiment, the hSBA strain is an LP2086 (fHBP) subfamily B strain that expresses a lipoprotein 2086 variant that is heterologous to strain CDC1127. In a preferred embodiment, the hSBA strain is an LP2086 (fHBP) subfamily B strain that expresses a lipoprotein 2086 variant that is heterologous to strain CDC1573.

In one embodiment, the immune response is bactericidal against a N. meningitidis serogroup B subfamily B strain that is heterologous to a N. meningitidis strain expressing B01. In one embodiment, the immune response is against N. meningitidis serogroup B B24 strain. In one embodiment, the immune response is against N. meningitidis serogroup B B44 strain. In one embodiment, the immune response is against N. meningitidis serogroup B B16 strain. In one embodiment, the immune response is against N. meningitidis serogroup B B03 strain. In one embodiment, the immune response is against N. meningitidis serogroup B B09 strain. In one embodiment, the immune response is against N. meningitidis serogroup B B15 strain. In one embodiment, the immune response is against N. meningitidis serogroup B B153 strain. In one embodiment, the immune response is bactericidal against a N. meningitidis serogroup B subfamily B strain that is heterologous to N. meningitidis strain CDC1573. In one embodiment, the immune response is bactericidal against a N. meningitidis serogroup B subfamily B strain that expresses a factor H binding protein including an amino acid sequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the second polypeptide. In another embodiment, the immune response is bactericidal against a N. meningitidis serogroup B subfamily B strain that expresses a factor H binding protein including an amino acid sequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a factor H binding protein expressed by N. meningitidis strain CDC1573. In a preferred embodiment, the immune response is bactericidal against a N. meningitidis serogroup B subfamily B strain that expresses a factor H binding protein including an amino acid sequence that has at least 80% identity, more preferably at least 87% identity, to a factor H binding protein expressed by N. meningitidis strain CDC1573. In another preferred embodiment, the immune response is bactericidal against a N. meningitidis serogroup B subfamily B strain that expresses a factor H binding protein including an amino acid sequence that has 100% identity to a factor H binding protein expressed by N. meningitidis strain CDC1573.

In another embodiment, the immune response is bactericidal against a N. meningitidis serogroup B subfamily B strain that expresses a factor H binding protein including an amino acid sequence that has at most 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the second polypeptide. In another embodiment, the immune response is bactericidal against a N. meningitidis serogroup B subfamily B strain that expresses a factor H binding protein including an amino acid sequence that has at most 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a factor H binding protein expressed by N. meningitidis strain CDC1573. In a preferred embodiment, the immune response is bactericidal against a N. meningitidis serogroup B subfamily B strain that expresses a factor H binding protein including an amino acid sequence that has at most 99% identity, more preferably at least 88% identity, to a factor H binding protein expressed by N. meningitidis strain CDC1573. Any minimum value may be combined with any maximum value described herein to define a range.

In one embodiment, the hSBA strain is an LP2086 (fHBP) B24 strain. In another embodiment, the hSBA strains is an LP2086 (fHBP) B44 strain. In a further embodiment, the hSBA strains includes LP2086 (fHBP) B24 and LP2086 (fHBP) B44 strains. In one embodiment, the hSBA strains includes LP2086 (fHBP) A22, LP2086 (fHBP) A56, LP2086 (fHBP) B24, and LP2086 (fHBP) B44 strains. In one embodiment, the hSBA strain includes B15. In one embodiment, the hSBA strain includes B153. In another embodiment, the hSBA strain is an LP2086 B16 strain. In one embodiment, the hSBA strain is an LP2086 B03 strain. In one embodiment, the hSBA strain is an LP2086 B09 strain. In a further embodiment, the hSBA strains include B24, B16, B44, B03, and B09, or any combination thereof. In another embodiment, the hSBA strains include B24, B16, B44, A22, B03, B09, A12, A19, A05, and A07, or any combination thereof. For example, in one embodiment, the hSBA strains include A22 and B24. In another embodiment, the hSBA strains include A06, A07, A12, A15, A19, A29, B03, B09, B15, and B16, or any combination thereof.

In one embodiment, the method induces an immune response against a N. meningitidis serogroup B subfamily A strain and against a N. meningitidis serogroup B subfamily B strain. Preferably, the immune response is bactericidal against a N. meningitidis serogroup B subfamily A strain and against a N. meningitidis serogroup B subfamily B strain.

In one embodiment, the immune response against the N. meningitidis serogroup B subfamily A strain is greater than the immune response against the N. meningitidis serogroup B subfamily B strain. For example, in one embodiment, the immunogenic composition induces higher bactericidal titers against a N. meningitidis serogroup B subfamily A strain than against a N. meningitidis serogroup B subfamily B strain, when tested under identical conditions. In one embodiment, the higher bactericidal titers against a N. meningitidis serogroup B subfamily A strain occurs within 30 days after a second dose of the immunogenic composition against N. meningitidis. In one embodiment, the higher bactericidal titers against a N. meningitidis serogroup B subfamily A strain occur in the absence of a third dose of the immunogenic composition against N. meningitidis.

In another embodiment, the immune response against the N. meningitidis serogroup B subfamily B strain is greater than the immune response against the N. meningitidis serogroup B subfamily A strain. For example, in one embodiment, the immunogenic composition induces higher bactericidal titers against a N. meningitidis serogroup B subfamily B strain than against a N. meningitidis serogroup B subfamily A strain, when tested under identical conditions. In one embodiment, the higher bactericidal titers against a N. meningitidis serogroup B subfamily B strain occurs within 30 days after a second dose of the immunogenic composition against N. meningitidis. In one embodiment, the higher bactericidal titers against a N. meningitidis serogroup B subfamily B strain occur in the absence of a third dose of the immunogenic composition against N. meningitidis.

Titers

In one embodiment, the composition induces an increase in bactericidal titer against N. meningitidis serogroup B in the human, as compared to the bactericidal titer against N. meningitidis serogroup B in the human prior to administration of a dose of the composition, when measured under identical conditions, e.g., in an hSBA. In one embodiment, the increase in bactericidal titer is compared to the bactericidal titer in the human before administration of the first dose of the composition, as compared to the bactericidal titer in the human prior to administration of the first dose of the composition, when measured under identical conditions, e.g., in an hSBA. In one embodiment, the increase in titer is observed after a second dose of the composition, as compared to the bactericidal titer in the human prior to administration of the second dose of the composition, when measured under identical conditions, e.g., in an hSBA. In another embodiment, the increase in bactericidal titer is observed after a third dose of the composition, as compared to the bactericidal titer in the human prior to administration of the third dose of the composition, when measured under identical conditions, e.g., in an hSBA.

In one embodiment, the composition induces a bactericidal titer against N. meningitidis serogroup B in the human after administration of a dose, wherein the bactericidal titer is at least greater than 1-fold higher than the bactericidal titer against N. meningitidis serogroup B in the human prior to administration of the dose, when measured under identical conditions, e.g., in an hSBA. For example, the bactericidal titer against N. meningitidis serogroup B may be at least 1.01-fold, 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, or 16-fold higher in the human after receiving a dose of the composition, as compared to the bactericidal titer against N. meningitidis serogroup B in the human prior to administration of the dose, when measured under identical conditions, e.g., in an hSBA.

In one embodiment, the composition when used in combination with an additional immunogenic compostion against human papillomavirus induces an increase in virucidal titer against human papillomavirus in the human, as compared to the virucidal titer against human papillomavirus in the human prior to administration of a dose of the composition, when measured under identical conditions. In one embodiment, the increase in virucidal titer is compared to the virucidal titer in the human before administration of the first dose of the composition, as compared to the virucidal titer in the human prior to administration of the first dose of the composition, when measured under identical conditions. In one embodiment, the increase in titer is observed after a second dose of the composition, as compared to the virucidal titer in the human prior to administration of the second dose of the composition, when measured under identical conditions. In another embodiment, the increase in virucidal titer is observed after a third dose of the composition, as compared to the virucidal titer in the human prior to administration of the third dose of the composition, when measured under identical conditions.

In one embodiment, the composition when used in combination with an additional immunogenic compostion against human papillomavirus induces a virucidal titer in the human after administration of a dose, wherein the virucidal titer against human papillomavirus is at least greater than 1-fold higher than the virucidal titer against human papillomavirus in the human prior to administration of the dose, when measured under identical conditions. For example, the virucidal titer against human papillomavirus may be at least 1.01-fold, 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, or 16-fold higher in the human after receiving a dose of the composition, as compared to the virucidal titer against human papillomavirus in the human prior to administration of the dose, when measured under identical conditions.

In one embodiment, a “responder” refers to a human, wherein the composition induces a bactericidal titer against N. meningitidis serogroup B in the human after administration of a dose, wherein the bactericidal titer against N. meningitidis serogroup B is at least greater than 1-fold higher than the bactericidal titer against N. meningitidis serogroup B in the human prior to administration of the dose. In a preferred embodiment, the responder achieves at least a 4-fold rise in hSBA titer, as compared to a bactericidal titer in the human prior to administration of the dose. Such a responder may be referred to as having a protective titer.

In one embodiment, the hSBA titer is the reciprocal of the highest dilution of a serum sample that produces a measurable effect. For example, in one embodiment, the hSBA titer is the reciprocal of the highest 2-fold dilution of a test serum that results in at least a 50% reduction of MnB bacteria (50% bacterial survival) compared to the T30 CFU value (i.e., the number of bacteria surviving after incubation in assay wells containing all assay components except test serum; 100% bacterial survival).

In one embodiment, the composition induces a bactericidal titer against N. meningitidis serogroup B in the human after receiving the first dose that is at least 2-fold higher than the bactericidal titer against N. meningitidis serogroup B in the human prior to receiving the first dose (e.g., higher than the bactericidal titer in the human in the absence of the first dose), when measured under identical conditions in the hSBA. In one embodiment, the composition induces a bactericidal titer against N. meningitidis serogroup B in the human that is at least 4-fold higher than the bactericidal titer against N. meningitidis serogroup B in the human prior to receiving the first dose, when measured under identical conditions in a human serum bactericidal assay that utilizes human complement (hSBA). In one embodiment, the composition induces a bactericidal titer against N. meningitidis serogroup B in the human that is at least 8-fold higher than the bactericidal titer against N. meningitidis serogroup B in the human prior to receiving the first dose, when measured under identical conditions in a human serum bactericidal assay that utilizes human complement (hSBA).

In a preferred embodiment, the human serum complement is derived from a human having low intrinsic bactericidal activity for a given SBA test strain. Low intrinsic bactericidal activity refers to, for example, a bactericidal titer that is at least less than a 1:4 dilution against the given SBA test strain. In one embodiment, the human complement is derived from a human having an hSBA titer that is at least less than 1:4, such as a 1:2 dilution, against the given SBA test strain, wherein the composition was not administered to the human.

A human may exhibit an hSBA titer of less than 1:4 prior to administration of a composition, such as the bivalent rLP2086 composition, or a human may exhibit an hSBA titer of ≥4 prior to administration of the composition. Accordingly, in preferred embodiments and examples, administration of at least one dose of the composition to the human results in an hSBA titer that is at least greater than 1:4, such as, for example, an hSBA titer of ≥8, an hSBA titer of ≥16, and an hSBA titer of ≥32. The respective Examples described herein include assessments of the proportion of human subjects having an hSBA titer ≥8 and/or ≥16, wherein the bivalent rLP2086 composition was administered to the human. Such preferred assessments of hSBA titers greater than 1:4 show that the protection, i.e., the bactericidal immune response induced in the human, is associated with the composition.

In one embodiment, the human has an hSBA titer equal to or greater than the hSBA's lower limit of quantitation (LLOQ) after administration of the first dose of the composition. In another embodiment, the human has an hSBA titer equal to or greater than the hSBA's LLOQ after administration of the second dose of the composition. In another embodiment, the human has an hSBA titer equal to or greater than the hSBA's LLOQ after administration of the third dose of the composition.

Additional Immunogenic Compositions

In one aspect, the invention relates to a method of inducing a bactericidal immune response against a N. meningitidis serogroup B subfamily A strain, and against a N. meningitidis serogroup B subfamily B strain in a human, in addition to inducing an immune response against at least one of any of the following: a N. meningitidis serogroup A strain, a N. meningitidis serogroup C strain, a N. meningitidis serogroup Y strain, and/or a N. meningitidis serogroup W135 strain; diphtheria, tetanus, and/or pertussis; hepatitis A virus; human papillomavirus; diphtheria, tetanus, pertussis and/or poliomyelitis; or any combination thereof.

The method includes administering to the human an effective amount of the immunogenic composition against N. meningitidis serogroup B, and concomitantly administering to the human at least one of the following immunogenic compositions: (i) an immunogenic composition against a N. meningitidis serogroup A strain, a N. meningitidis serogroup C strain, a N. meningitidis serogroup Y strain, and/or a N. meningitidis serogroup W135 strain; (ii) an immunogenic composition against diphtheria, tetanus, and pertussis; (iii) an immunogenic composition against hepatitis A virus; (iv) an immunogenic composition against human papillomavirus; (v) an immunogenic composition against diphtheria, tetanus, pertussis and poliomyelitis; or (vi) any combination thereof.

In one embodiment, the method includes administering to the human an effective amount of the immunogenic composition against N. meningitidis serogroup B, and concomitantly administering to the human at least one of the following immunogenic compositions: (i) MENACTRA, MENVEO, or NIMENRIX; (ii) ADACEL; (iii) HAVRIX; (iv) GARDASIL; (v) REPEVAX; or (vi) any combination thereof.

For example, in exeplary embodiments, the method includes concomitantly administering to the human at least one of the following combination of compositions:

Exemplary The rLP2086 MCV4 Tdap HAV HPV dTaP composition: composition composition composition composition composition composition Combination 1 rLP2086 MCV4 Tdap HAV HPV dTaP composition composition composition composition composition composition Combination 2 rLP2086 Tdap HAV HPV dTaP composition composition composition composition composition Combination 3 rLP2086 MCV4 HAV HPV dTaP composition composition composition composition composition Combination 4 rLP2086 MCV4 Tdap HPV dTaP composition composition composition composition composition Combination 5 rLP2086 MCV4 Tdap HAV dTaP composition composition composition composition composition Combination 6 rLP2086 MCV4 Tdap HAV HPV composition composition composition composition composition Combination 7 rLP2086 MCV4 Tdap HAV composition composition composition composition Combination 8 rLP2086 MCV4 HAV HPV composition composition composition composition Combination 9 rLP2086 MCV4 HPV dTaP composition composition composition composition Combination 10 rLP2086 MCV4 Tdap dTaP composition composition composition composition Combination 11 rLP2086 Tdap HAV HPV composition composition composition composition Combination 12 rLP2086 Tdap HPV dTaP composition composition composition composition Combination 13 rLP2086 Tdap HAV dTaP composition composition composition composition Combination 14 rLP2086 HAV HPV dTaP composition composition composition composition Combination 15 rLP2086 MCV4 HAV dTaP composition composition composition composition Combination 16 rLP2086 MCV4 Tdap HPV composition composition composition composition Combination 17 rLP2086 MCV4 Tdap composition composition composition Combination 18 rLP2086 MCV4 HAV composition composition composition Combination 19 rLP2086 MCV4 HPV composition composition composition Combination 20 rLP2086 MCV4 dTaP composition composition composition Combination 21 rLP2086 Tdap HAV composition composition composition Combination 22 rLP2086 Tdap HPV composition composition composition Combination 23 rLP2086 Tdap dTaP composition composition composition Combination 24 rLP2086 HAV HPV composition composition composition Combination 25 rLP2086 HAV dTaP composition composition composition Combination 26 rLP2086 HPV dTaP composition composition composition Combination 27 rLP2086 MCV4 composition composition Combination 28 rLP2086 Tdap composition composition Combination 29 rLP2086 HAV composition composition Combination 30 rLP2086 HPV composition composition Combination 31 rLP2086 dTaP composition composition Combination 32 rLP2086 MENACTRA, composition MENVEO, or NIMENRIX Combination 33 rLP2086 ADACEL composition Combination 34 rLP2086 HAVRIX composition Combination 35 rLP2086 GARDASIL composition Combination 36 rLP2086 REPEVAX composition

“Concomitant” and “concomitantly” and “coadministered” and “coadministration” as used herein refer to the administration of at least two compositions to a human simultaneously or within a time period during which the effects of the first administered composition is still effective in the human. If the first composition is, e.g., the rLP2086 composition and the second composition is an MCV4 composition, the concomitant administration of the second composition can occur within one to seven days, preferably within 48 hours, more preferably within 24 hours, of the administration of the first composition. In one embodiment, the concomitant administration occurs within at most 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 24 hours, or 48 hours of the administration of the first composition. In another embodiment, the concomitant administration occurs simultaneously to administration of the first composition.

In a concomitant administration, the first composition may be administered before, after, or simultaneously to the additional composition(s) within the specified time period. For example, in one embodiment, the first composition, e.g., the rLP2086 composition, is administered to the human first and the second composition, e.g., an MCV4 composition, is administered within 24 hours after the rLP2086 composition. In another embodiment, the first composition, e.g., an MCV4 composition, is administered to the human first and the second composition, e.g., the rLP2086 composition, is administered to the human within 24 hours after the MCV4 composition.

In one embodiment, the additional composition(s) is administered before, after, or simultaneously to the first dose of the rLP2086 composition within the specified time period. In another embodiment, In one embodiment, the additional composition(s) is administered before, after, or simultaneously to the second dose of the rLP2086 composition within the specified time period. In a further embodiment, the additional composition(s) is administered before, after, or simultaneously to the third dose of the rLP2086 composition within the specified time period. In yet another embodiment, the additional composition(s) is administered before, after, or simultaneously to a booster dose of the rLP2086 composition within the specified time period.

The inventors surprisingly discovered that the rLP2086 composition may be administered with at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 additional antigens without negatively affecting the immune response against N. meningitidis. For example, Example 11 demonstrates that substantial hSBA responses to N. meningitidis strains and to each of the at least 10 MCV4 and Tdap antigens were observed. Example 13 demonstrates that the rLP2086 composition may be coadministered with the 14 antigens included amongst HPV4, MCV4, and Tdap vaccines while inducing an effective immune response, which may include a bactericidal and/or virucidal immune response.

Accordingly, in one aspect, the invention relates to a method of inducing an immune response against a N. meningitidis serogroup A strain, a N. meningitidis serogroup C strain, a N. meningitidis serogroup Y strain, a N. meningitidis serogroup W135 strain, and a N. meningitidis serogroup B strain. In a preferred embodiment, the immune response comprises a bactericidal immune response. The method includes administering to the human an effective amount of a N. meningitidis rLP2086 composition, said composition including a) a first lipidated polypeptide having the amino acid sequence set forth in SEQ ID NO: 1, and b) a second lipidated polypeptide having the amino acid sequence set forth in SEQ ID NO: 2; and a further composition including a mixture of four distinct and separately made protein-capsular polysaccharide conjugates, wherein the first conjugate includes a purified N. meningitidis capsular polysaccharide of serogroup W-135 conjugated to a carrier protein, the second conjugate includes a purified N. meningitidis capsular polysaccharide of serogroup Y conjugated to a carrier protein, the third conjugate includes a purified N. meningitidis capsular polysaccharide of serogroup A conjugated to a purified carrier protein, and the fourth conjugate includes a purified N. meningitidis capsular polysaccharide of serogroup C conjugated to a carrier protein, wherein the carrier protein is selected from the group consisting of diphtheria toxoid, CRM197, and tetanus toxoid.

In one preferred embodiment, the method includes administering an MCV4 composition, wherein the carrier protein is diphtheria toxoid. In one embodiment, the method includes administering MENACTRA vaccine.

In another embodiment, the method includes administering an MCV4 composition wherein the carrier protein is CRM₁₉₇. In one embodiment, the method includes administering MENVEO vaccine. MENVEO [Meningococcal (Groups A, C, Y and W-135) Oligosaccharide Diphtheria CRM197 Conjugate Vaccine] is a sterile liquid vaccine administered by intramuscular injection that contains N. meningitidis serogroup A, C, Y and W-135 oligosaccharides conjugated individually to Corynebacterium diphtheriae CRM₁₉₇ protein.

In yet another embodiment, the method includes administering an MCV4 composition wherein the carrier protein is tetanus toxoid. In one embodiment, the method includes administering NIMENRIX vaccine. NIMENRIX® (meningococcal polysaccharide groups A, C, W-135 and Y conjugate vaccine) is a tetravalent meningococcal polysaccharide conjugated vaccine including N. meningitidis capsular polysaccharides A, C, W-135 and Y each coupled to tetanus toxoid as a carrier protein. The N. meningitidis serogroups A and C polysaccharides are conjugated with an adipic dihydrazide (AH) spacer and indirectly conjugated to the tetanus toxoid whereas the W-135 and Y polysaccharides are conjugated directly to tetanus toxoid.

In one embodiment, the method increases titers against N. meningitidis serogroup A, C, W-135, and/or Y at least greater than 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, or more, as compared to the titers in the human prior to administration of the immunogenic compositions. In another embodiment, the method increases titers against N. meningitidis serogroup A, C, W-135, and Y at least greater than 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, or more, as compared to the titers in the human prior to administration of the immunogenic compositions. In another embodiment, the method increases titers against N. meningitidis serogroup A, C, W-135, and Y and serogroup B at least greater than 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, or more, as compared to the titers in the human prior to administration of the immunogenic compositions.

In another aspect, the invention relates to a method of inducing an immune response against a N. meningitidis serogroup B strain and against tetanus, diphtheria, and pertussis in a human. In a preferred embodiment, the immune response comprises a bactericidal immune response. In another preferred embodiment, the immune response comprises a virucidal immune response. In another preferred embodiment, the immune response comprises a bactericidal and virucidal immune response. The method includes administering to the human an effective amount of a N. meningitidis rLP2086 composition, said composition including a) a first lipidated polypeptide having the amino acid sequence set forth in SEQ ID NO: 1, and b) a second lipidated polypeptide having the amino acid sequence set forth in SEQ ID NO: 2; and a further composition including tetanus and diphtheria toxoids and pertussis antigens, preferably adsorbed on aluminum phosphate. In one embodiment, the method includes administering ADACEL vaccine.

In one embodiment, the method increases titers against tetanus, diphtheria, and and/or pertussis at least greater than 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, or more, as compared to the titers in the human prior to administration of the immunogenic compositions. In another embodiment, the method increases titers against tetanus, diphtheria, and pertussis at least greater than 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, or more, as compared to the titers in the human prior to administration of the immunogenic compositions. In another embodiment, the method increases titers against tetanus, diphtheria, and pertussis, and N. meningitidis serogroups A, C, W-135, and Y and serogroup B at least greater than 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, or more, as compared to the titers in the human prior to administration of the immunogenic compositions.

In one aspect, the invention relates to a method of inducing an immune response against a N. meningitidis serogroup B strain, against N. meningitidis serogroup A, C, Y, and W135 strains, and against tetanus, diphtheria, and pertussis in a human. In a preferred embodiment, the immune response comprises a bactericidal immune response. In another preferred embodiment, the immune response comprises a virucidal immune response. In another preferred embodiment, the immune response comprises a bactericidal and virucidal immune response. In one embodiment, the method includes concomitantly administering the rLP2086 composition, MENACTRA, and ADACEL vaccines.

In one aspect, the invention relates to a method of inducing an immune response against a N. meningitidis serogroup B strain, against N. meningitidis serogroups A, C, Y, and W135 strains, and against tetanus, diphtheria, and pertussis, and further against against human papillomavirus in a human. In a preferred embodiment, the immune response comprises a bactericidal immune response. In another preferred embodiment, the immune response comprises a virucidal immune response. In another preferred embodiment, the immune response comprises a bactericidal and virucidal immune response. In one embodiment, the method includes concomitantly administering the rLP2086 composition, MENACTRA, and ADACEL vaccines. In one embodiment, the method includes concomitantly administering the rLP2086 composition, MENACTRA, ADACEL, and GARDASIL vaccines.

The inventors surprisingly discovered that the immunogenic composition against N. meningitidis may be administered with an immunogenic composition against human papillomavirus (HPV) without negatively affecting the bactericidal response against N. meningitidis. As explained in Example 7 and Example 8, substantial hSBA responses to N. meningitidis test strains were observed among humans who were administered with the immunogenic composition against N. meningitidis and GARDASIL and in humans who were administered with the immunogenic composition against N. meningitidis and saline. Additional increases in hSBA responses were observed about 1 month after a third dose of the immunogenic composition against N. meningitidis.

Moreover, the inventors surprisingly discovered that robust immune responses against both N. meningitidis and HPV were generated in the human following an administration of both the immunogenic composition against N. meningitidis and the immunogenic composition against HPV, as compared to the immune response in the human before administration of the compositions. As explained in Example 7 and Example 8, titers against HPV increased in the human after an administration of the immunogenic composition against N. meningitidis and GARDASIL, as compared to the titers in the human prior to administration of the immunogenic compositions. The increase in titers against HPV was at least greater than 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, or more.

Accordingly, in one embodiment, the method includes inducing an immune response against N. meningitidis in a human, wherein the method further includes administering to the human an immunogenic composition against human papillomavirus. Preferably, the immune response is bactericidal against N. meningitidis. In one embodiment, the method further includes inducing an immune response against HPV. In a preferred embodiment, the method further includes inducing an immune response against at least one of human papillomavirus types 6, 11, 16, and 18, or any combination thereof. In one embodiment, the immunogenic composition against HPV is administered to the human within 24 hours of administering said composition against N. meningitidis.

In one embodiment, the method includes inducing an immune response against N. meningitidis in a human, wherein the method further includes administering to the human an immunogenic composition against HPV. Preferably, the immune response is bactericidal against N. meningitidis. In one embodiment, the method further includes inducing an immune response against HPV. In a preferred embodiment, the method further includes inducing an immune response against at least one of human papillomavirus types 6, 11, 16, and 18, or any combination thereof. In one embodiment, the immunogenic composition against human papillomavirus is administered to the human within 24 hours of administering said composition against N. meningitidis.

In another aspect, the inventors surprisingly discovered that the immunogenic composition against N. meningitidis may be administered with an immunogenic composition against diphtheria, tetanus, acellular pertussis, and inactivated poliomyelitis virus (dTaP) without negatively affecting the bactericidal response against N. meningitidis. As explained in Example 4, substantial hSBA responses to N. meningitidis test strains were observed among humans who were administered with the immunogenic composition against N. meningitidis and REPEVAX. Additional increases in hSBA responses were observed about 1 month after a third dose of the immunogenic composition against N. meningitidis.

Moreover, the inventors surprisingly discovered that robust immune responses against both N. meningitidis and dTaP were generated in the human following an administration of both the immunogenic composition against N. meningitidis and the immunogenic composition against dTaP, as compared to the immune response in the human before administration of the compositions. As explained in Example 4, titers against dTaP increased in the human after an administration of the immunogenic composition against N. meningitidis and REPEVAX, as compared to the titers in the human prior to administration of the immunogenic compositions. The increase in titers against dTaP was at least greater than 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, or more.

Methods and Administration

In one aspect, the invention relates to a method of inducing an immune response in a human. In a preferred embodiment, the immune response comprises a bactericidal immune response. In another preferred embodiment, the immune response comprises a virucidal immune response. In another preferred embodiment, the immune response comprises a bactericidal and virucidal immune response. In another preferred embodiment, the immune response comprises inactivating the bacteria and/or virus.

In another aspect, the invention relates to a method of vaccinating a human. In one embodiment, the method includes administering to the human at least one dose of the composition described above. In another embodiment, the method includes administering to the human at least a first dose and a second dose of the composition described above.

Surprisingly, the inventors discovered that a two-dose schedule of the rLP2086 composition induced a bactericidal titer against diverse heterologous subfamily A and against diverse heterologous subfamily B strains in the human. For example, the percentage of humans with an hSBA titer ≥8 was 90% or greater for SBA test strains expressing LP2086 (fHBP) A22 or LP2086 (fHBP) A56 following a two-dose schedule of the composition described above. See Example 1.

In one embodiment, the second dose is administered at least 20, 30, 50, 60, 100, 120, 160, 170, or 180 days after the first dose, and at most 250, 210, 200, or 190 days after the first dose. Any minimum value may be combined with any maximum value described herein to define a range.

In another embodiment, the second dose is administered about 30 days after the first dose. In another embodiment, the second dose is administered about 60 days after the first dose, such as, for example, in a 0, 2 month immunization schedule. In another embodiment, the second dose is administered about 180 days after the first dose, such as, for example, in a 0, 6 month immunization schedule. In yet another embodiment, the second dose is administered about 120 days after the first dose, such as, for example, in a 2, 6 month immunization schedule.

In one embodiment, the method includes administering to the human two doses of the composition and at most two doses. In one embodiment, the two doses are administered within a period of about 6 months after the first dose. In one embodiment, the method does not include further administration of a booster to the human. A “booster” as used herein refers to an additional administration of the composition to the human. Administering to the human at most two doses of the composition may be advantageous. Such advantages include, for example, facilitating a human to comply with a complete administration schedule and facilitating cost-effectiveness of the schedule.

In one embodiment, the first dose and the second dose are administered to the human over a period of about 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 days, and most 400, 390, 380, 370, 365, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, or 200 days after the first dose. Any minimum value may be combined with any maximum value described herein to define a range.

In one embodiment, the first dose and the second dose are administered to the human over a period of about 30 days. In another embodiment, the first dose and the second dose are administered to the human over a period of about 60 days. In another embodiment, the first dose and the second dose are administered to the human over a period of about 180 days.

Three Doses

The inventors further surprisingly discovered that a three-dose schedule of the rLP2086 composition induced a broader bactericidal titer against strains expressing heterologous LP2086 (fHBP) subfamily B strains in a greater percentage of humans than a two-dose schedule. For example, the percentage of humans with a hSBA titer ≥8 was 65% or greater for SBA test strains LP2086 (fHBP) B24 and LP2086 (fHBP) B44 following a two-dose schedule of the composition described above. The percentage of humans with a hSBA titer ≥8 was 86% or greater for SBA test strains B24 and B44 following a three-dose schedule of the composition described above. See Example 1.

Accordingly, in one embodiment, a three-dose schedule of the composition induces a bactericidal titer against multiple strains expressing LP2086 (fHBP) heterologous to the first and/or second polypeptide in a greater percentage of humans than a two-dose schedule.

In one embodiment, the method includes administering to the human three doses of the composition. In another embodiment, the method includes administering at most three doses of the composition. In one embodiment, the three doses are administered within a period of about 6 months after the first dose. In one embodiment, the method includes an administration of a booster dose to the human after the third dose. In another embodiment, the method does not include administration of a booster dose to the human after the third dose. In another embodiment, the method does not further include administering a fourth or booster dose of the composition to the human. In a further embodiment, at most three doses within a period of about 6 months are administered to the human.

In an exemplary embodiment, the second dose is administered about 30 days after the first dose, and the third dose is administered about 150 days after the second dose, such as, for example, in a 0, 1, 6 month immunization schedule. In another exemplary embodiment, the second dose is administered about 60 days after the first dose, and the third dose is administered about 120 days after the second dose, such as, for example, in a 0, 2, 6 month immunization schedule.

In one embodiment, the first dose, second dose, and third dose are administered to the human over a period of about 150, 160, 170, or 180 days, and at most 240, 210 200, or 190 days. Any minimum value may be combined with any maximum value described herein to define a range. Preferably, the first dose, second dose, and third dose is administered to the human over a period of about 180 days or 6 months. For example, the second dose may be administered to the human about 60 days after the first dose, and the third dose may be administered to the human about 120 days after the second dose. Accordingly, an exemplary schedule of administration includes administering a dose to the human at about months 0, 2, and 6.

As described above, multiple doses of the immunogenic composition may be administered to the human, and the number of days between each dose may vary. An advantage of the method includes, for example, flexibility for a human to comply with the administration schedules.

EXAMPLES

The following Examples illustrate embodiments of the invention. Unless noted otherwise herein, reference is made in the following Examples to an investigational bivalent recombinant vaccine (rLP2086), which is a preferred exemplary embodiment of a composition including 60 μg of a first lipidated polypeptide including the amino acid sequence set forth in SEQ ID NO: 1 per 0.5 mL dose, 60 μg of a second lipidated polypeptide including the amino acid sequence set forth in SEQ ID NO: 2 per 0.5 mL dose, 2.8 molar ratio polysorbate-80 to the first polypeptide, 2.8 molar ratio polysorbate-80 to the second polypeptide, 0.5 mg Al³⁺/ml of the composition, 10 mM histidine, and 150 mM sodium chloride. More specifically, the investigational bivalent recombinant rLP2086 vaccine includes (a) 60 μg of a first lipidated polypeptide including the amino acid sequence set forth in SEQ ID NO: 1; (b) 60 μg of a second lipidated polypeptide including the amino acid sequence set forth in SEQ ID NO: 2; (c) 18 μg polysorbate-80; (d) 250 μg aluminum; (e) 780 μg histidine, and (f) 4380 μg sodium chloride. Each dose was 0.5 mL.

Example 1: Safety, Tolerability, and Immunogenicity of an Investigational Meningococcal Serogroup B Bivalent (MnB) rLP2086 Vaccine in Healthy Adolescents When Administered in Regimens of 2 or 3 Doses in Healthy Subjects Aged 11 to 18 Years

Background: Safety, tolerability, and immunogenicity of an investigational bivalent, recombinant vaccine (rLP2086) were studied in healthy adolescents 11-18 years of age using 5 dose regimens including 2 or 3 vaccinations (Table 1).

The vaccine is a 0.5 ml-dose formulated to contain 60 μg each of a purified subfamily A and a purified subfamily B rLP2086 protein, 2.8 molar ratio polysorbate-80, and 0.25 mg of Al³⁺ as AlPO₄, 10 mM histidine-buffered saline at pH 6.0.

Saline is used as a placebo because there is no known proven safe, immunogenic, and effective vaccine against MnB that could serve as an active control. The normal saline solution includes 0.9% sodium chloride in a 0.5 ml dose.

Methods: All subjects in this phase 2, randomized, placebo-controlled, single-blind study attended vaccination visits at months 0, 1, 2 and 6. For blinding, a saline control was given when vaccine was not scheduled. Serum bactericidal assays using human complement (hSBA) were performed with 4 MnB test strains expressing LP2086 (fHBP) fHBP variants A22, A56, B24 and B44 (i.e., the 4 “primary hSBA test strains” in the primary endpoint analysis), all of which are different from the variants in the vaccine. Unsolicited adverse events (AE), solicited local and systemic reactions, and antipyretic use were assessed.

Geometric mean hSBA titers were computed for each primary strain at each blood sampling time point along with 2-sided 95% confidence intervals (Cis). Geometric mean fold rises were computed along with 95% Cls.

A responder was defined as a subject with an hSBA titer equal or above the lower limit of quantitation (LLOQ) of the hSBA assays. The LLOQ for each of the 4 hSBA test strains in the primary endpoint analysis was an hSBA titer equal to 1:8. The limit of detection (LOD) for each primary test strain was a titer equal to 1:4 (widely viewed as the correlate of protection against meningococcal disease).

Results: 1 month after the last vaccine dose, 86-99% subjects (after 3 doses; P<0.001) and 69-100% of subjects (after 2 doses) had hSBA titers to each MnB test strain. After study dose 1, 19-27% (1.1-4.3% severe) and 23-27% (0.0-1.0% severe) of rLP2086 recipients experienced redness and swelling, respectively, by group. Injection site pain was the most common local reaction after study dose 1 (7.6-13.1% severe). Fever 38° C. after the first study dose of the bivalent rLP2086 vaccine was experienced in 3.3-6.5% by group compared to 2.1% in saline recipients. Local and systemic reactions were generally more frequent after dose 1 than after subsequent doses. 43 of 1712 subjects (2.5%) reported 51 serious AEs; 2 cases were considered related (1 case of vertigo, chills and headache and 1 case of fever and vomiting). No deaths were reported.

TABLE 1 Table. Statistical Analysis on Proportion of Evaluable Study Subjects Achieving hSBA Titer ≥8* for Each Primary Strain 1 Month After Last Dose of Bivalent rLP2086 - Evaluable Immunogenicity Population Group 1 Group 2 Group 3 Group 4 Group 5 (0, 1, 6 mo) (0, 2, 6 mo) (0, 6 mo) (0, 2 mo) (2, 6 mo) Strain (95% (95% (95% (95% (95% [variant] n^(†)/N^(‡) %^(§) CI)^(¶) n^(†)/N^(‡) %^(§) CI)^(¶) n^(†)/N^(‡) %^(§) CI)^(¶) n^(†)/N^(‡) % CI)^(¶) n^(†)/N^(‡) % CI)^(¶) PMB80 330/360 91.7^(¶) (88.3, 339/357 95.0^(¶) (92.1, 345/369 93.5^(¶) (90.5, 216/238 90.8 (86.3, 102/111 91.9 (85.2, [A22] 94.3) 97.0) 95.8) 94.1) 96.2) PMB2001 360/362 99.4^(¶) (98.0, 355/359 98.9^(¶) (97.2, 364/370 98.4^(¶) (96.5, 240/240 100.0  (98.5, 112/113 99.1 (95.2, [A56] 99.9) 99.7) 99.4) 100.0) 100.0) PMB2948 315/354 89.0^(¶) (85.2, 313/354 88.4^(¶) (84.6, 291/359 81.1^(¶) (76.6, 173/237 73.0 (66.9,  76/110 69.1 (59.6, [B24] 92.0) 91.6) 85.0) 78.5) 77.6) PMB2707 315/356 88.5^(¶) (84.7, 303/352 86.1^(¶) (82.0, 276/356 77.5^(¶) (72.2, 164/234 70.1 (63.8,  81/111 73.0 (63.7, [B44] 91.6) 89.5) 82.3) 75.9) 81.0) *Lower limit of quantification for all strains = 8. ^(†)Number of subjects with hSBA titer ≥8. ^(‡)Number of subjects with valid hSBA titers. ^(§)P < 0.001 using one-sided exact test based on binomial distribution; values <0.0125 are considered significant. ^(¶)Exact 2-sided confidence interval (Clopper and Pearson) based upon the observed proportion of subjects.

Conclusions: Bivalent rLP2086 had an acceptable safety profile. All 5 dosing regimens yielded hSBA titers against all 4 test strains in a high proportion of subjects. The higher proportions against some test strains after 3 doses compared with 2 doses indicate that 3 doses may provide the broadest protection against diverse MnB clinical strains. Global phase 3 clinical trials are underway with the bivalent rLP2086 vaccine.

One of the objectives of this study was to assess the immune response, as measured by hSBA performed with MnB strains expressing LP2086 subfamily A and B proteins, 1 month after the third vaccination with bivalent rLP2086, among Group 1 subjects (0-, 1-, and 6-month schedule as randomized) and among Group 2 subjects (0-, 2-, and 6-month schedule as randomized). An endpoint for the immunogenicity analysis was the proportion of subjects in Groups 1 and 2 achieving an hSBA titer LLOQ at Month 7 (or 1 month after the third dose of bivalent rLP2086) for each of the 4 primary MnB test strains (A22, A56, B24, and B44). The LLOQ was 1:8 for the 4 primary MnB test strains.

For the evaluable immunogenicity population, the proportion of subjects in Group 1 achieving an hSBA titer 1:8 after 3 doses of bivalent rLP2086 was 91.7% for A22, 99.4% for A56, 89% for B24, and 88.5% for B44 (See Table 1 above). Since the lower limit of the 97.5% CI was >50% for all strains (87.8%, p<0.001; 97.8%, p<0.001; 84.7%, p<0.001; and 84.1%, p<0.001 for strains A22, A56, B24, and B44, respectively, the study objective was met for subjects in Group 1.

For Group 2, the proportion of subjects achieving an hSBA titer 1:8 after 3 doses of bivalent rLP2086 was 95.0% for A22, 98.9% for A56, 88.4% for B24, and 86.1% for B44 (See Table 1 above). Similar to what was seen for Group 1, the lower limit of the 97.5% CI was >50% for all strains (91.7%, p<0.001; 96.9%, p<0.001; 84.1%, p<0.001; and 81.4%, p<0.001 for strains A22, A56, B24, and B44, respectively, demonstrating that the objective was also met for the subjects in Group 2.

A secondary objective was to assess the immune response, as measured by hSBA performed with MnB strains expressing LP2086 subfamily A and B proteins, 1 month after the second dose of bivalent rLP2086, among group 3 subjects (0- and 6-month schedule as randomized). This secondary objective was the proportion of subjects in Group 3 achieving an hSBA titer ≥LLOQ (1:8) at Month 7 (or 1 month after the second dose of bivalent rLP2086) for each of the 4 primary MnB test strains.

This secondary objective was also met since the proportion of subjects in Group 3 achieving an hSBA titer 1:8 after 2 doses of bivalent rLP2086 was 93.5%, 98.4%, 81.1%, and 77.5% for the primary MnB test strains with the lower limit of the 97.5% CI>50% for all strains (90.0%, p<0.001; 96.2%, p<0.001; 76.0%, p<0.001; and 72.2%, p<0.001 for strains A22, A56, B24, and B44, respectively. See Table 1 above).

Another secondary objective was the proportion of subjects with hSBA titer LLOQ for each of the 4 primary MnB test strains at each blood sampling time point for subjects in Groups 1 to 5. The LLOQ for each of the 4 primary hSBA test strains was a titer of 1:8. The proportions of subjects with an hSBA titer 1:8 by study time for the evaluable immunogenicity population is shown in Table 1 above.

The proportion of subjects who had an hSBA titer 1:8 after 1 dose of bivalent rLP2086 (Group 5 [2- and 6-month schedule] 1 month after Injection 3) was 55.9% for A22, 67.6% for A56, 56.9% for B24, and 23.8% for B44.

The proportion of subjects who had an hSBA titer 1:8 one month after 2 doses of bivalent rLP2086 ranged from 74.6% to 100% for subfamily A strains, and from 54.0% to 81.1% for subfamily B strains. After 3 doses, the proportion increased and ranged from 91.7% to 99.4% and from 86.1% to 89.0% for subfamily A and B strains, respectively.

Example 2: Serum Bactericidal Assay Using Human Complement (HSBA)

MnB clearance from the human bloodstream is primarily achieved by complement-mediated bacteriolysis and an intact complement system is important for resistance against infections caused by MnB. The in vivo complement-mediated bacteriolysis of MnB is mimicked in vitro by the serum bactericidal assay using human complement (hSBA), a functional serological assay shown to be the surrogate of protection for meningococcal disease. That is, demonstration of bacterial killing in the serum bactericidal assay using human complement (hSBA) correlates with protection against meningococcal disease. Immunity elicited by the vaccine is determined using hSBAs against 4 MnB strains (fHBP variants A22, A56, B24, and B44).

The four primary MnB test strains were used in the hSBAs described in the Examples for the determination of endpoints. That is, these strains were used to estimate vaccine efficacy using hSBA immunogenicity endpoints. These test strains represent 4 of the 6 fHBP phylogenetic subgroups that account for >90% of disease isolates circulating in the USA and Europe.

On 29 Oct. 2014, TRUMENBA® (bivalent recombinant lipoprotein 2086 [rLP2086]) was approved in the United States to prevent invasive meningococcal disease caused by MnB in individuals aged 10 to 25 years. Approval of TRUMENBA® was based on demonstration of immune response, as measured by serum bactericidal activity against four serogroup B strains representative of IMD strains in the United States and Europe.

Identity to matched fHBP subfamily vaccine fHBP Lipooligosaccharides Variant component subgroup CC PorA Sialation Level (mol %) A56 98.1% N1C2 CC213 P1.22,14 55% B44 91.6% N4/N5 CC269 P1.19- 23% 1,10-4 A22 88.9% N2C2 CC41/44 P1.21,16 84% B24 86.2% N6 CC32 P1.12- 22% 1,13-1

In selecting the 4 primary MnB test strains from invasive disease isolates, an approach was used which took into account the population distribution of the in vitro LP2086 surface expression. Furthermore, the hSBA test strains had to show low baseline hSBA positivity, as the populations at risk for meningococcal disease are characterized by non-existing or low baseline bactericidal activity to most strains. In addition, each of the 4 primary MnB test strains expresses an LP2086 variant that is different from the LP2086 variant in the vaccine, thus allowing an objective assessment of functional immunogenicity and efficacy to invasive meningococcal disease (IMD) strains circulating in the population.

The hSBA measures the amount of anti-meningococcal serogroup B (MnB) antibody in serum capable of initiating complement-mediated bactericidal activity. Briefly, test serum is serially-diluted in 2-fold steps and added to 96-well assay plates. MnB SBA test strains and human serum complement are added, initiating the bactericidal reaction. After incubation of the assay plates at 37° C. for 30-60 minutes (depending on SBA test strain; called T30), the reaction mixture containing bacteria surviving this incubation are diluted and transferred to microfilter plates. Following overnight incubation, surviving bacteria expressed as colony-forming units (CFU) are enumerated using an Immunospot Analyzer. The raw CFU data are recorded electronically and transferred to a data analysis application that calculates the hSBA titer. The hSBA titer is the reciprocal of the highest 2-fold dilution of a test serum that results in at least a 50% reduction of MnB bacteria (50% bacterial survival) compared to the T30 CFU value (i.e., the number of bacteria surviving after incubation in assay wells containing all assay components except test serum; 100% bacterial survival). Titers may be reported as step titers, i.e., 1:4, 1:8, 1:16, etc. Serum samples are tested by two individual, replicate determinations in the same assay. The final titer reported for samples in which the replicate measurements are not identical is the lower of the two replicate measurements when system suitability and sample suitability criteria (e.g. replicate titers must agree within one 2-fold dilution) are met.

hSBA assays were done after serially diluting test sera in Dulbecco's phosphate-buffered saline. Bacteria (roughly 2000 colony-forming units) and human serum complement (20% by weight final concentration) were added to the serially diluted sera in 96-well plates and incubated at 37° C. for 30-40 min (depending on hSBA test strain) in a small-radius orbital shaker at 700 rpm. After incubation, a portion of the reaction mixture was transferred to microfilter plates. After overnight incubation, surviving bacteria were counted with an Immunospot Analyzer (Cellular Technology Limited; Shaker Heights, Ohio, USA) and hSBA titers were analysed with SAS (version 9.2). The hSBA titer was calculated as the reciprocal of the interpolated test serum dilution that resulted in a 50% reduction of bacteria compared with a control not subjected to test serum (i.e., surviving bacteria at the end of the hSBA reaction). Per protocol hSBAs were done on the basis of the hSBA titer that was at or above the lower limit of quantitation of the hSBA assays as established during qualification of the assays with strains listed in the Table 1 of Example 1.

Human serum is the complement source for the SBA. However, the hSBA titers may vary depending on the human complement lot used. Accordingly, human complement is preferably controlled through rigorous screening and qualification to ensure consistent performance in the hSBA. For the hSBA, human serum complement may be pooled from multiple normal healthy human adults or used from individual donors (i.e., not pooled).

Example 3—Polysorbate-80

Three parameters have been optimized for drug product formulation: pH, aluminum concentration and polysorbate 80 (PS-80) to protein molar ratio. In a dose of the composition having a total volume of 0.5 ml, optimal protein binding to aluminum is achieved at a pH of about 6.0 and about a 0.5 mg/ml concentration of aluminum as aluminum phosphate (AlPO₄) (which is equivalent to 0.25 mg aluminum per dose). The PS-80 to protein molar ratio is maintained at 2.8±1.4 in order to stabilize the formulation with respect to in vitro potency. Polysorbate 80 (PS-80) is added to drug substance to obtain the target PS-80 to protein molar ratio of 2.8. Therefore, PS-80 is preferably not added during the drug product formulation.

Example 4

Randomized, Placebo-Controlled, Phase 2 Study of the Immunogenicity and Safety of REPEVAX® Administered Concomitantly With Bivalent rLP2086 Vaccine in Healthy Adolescents

Background/Aims: The investigational bivalent rLP2086 vaccine, being developed to prevent Neisseria meningitidis serogroup B (MnB) disease in adolescents, was evaluated with concomitant administration of REPEVAX®, a dTaP-inactivated polio vaccine (which may be described in U.S. Pat. No. 7,479,283, WO1990/013313, and EP1666057 B1, and UK Marketing Authorisation PL06745/0121) currently used in this population.

Methods: Adolescents, randomized 1:1 to REPEVAX+rLP2086 or REPEVAX+saline were vaccinated at 0, 2, and 6 months. The proportion of subjects achieving prespecified antibody levels to 9 REPEVAX antigens 30 days after initial vaccination were determined. Immune responses (hSBA) to 4 MnB test strains were measured 30 days after vaccinations 2 and 3. Adverse events (AE) and local/systemic reactions were assessed.

REPEVAX (Sanofi Pasteur MSD limited) is a combined low-dose diphtheria, tetanus, acellular pertussis, and inactivated poliomyelitis virus vaccine containing diphtheria toxoid (not less than 2 IU), tetanus toxoid (not less than 20 IU), pertussis antigens (pertussis toxoid (2.5 micrograms), filamentous haemagglutinin (5 micrograms), pertacti (3 micrograms), and fimbriae Types 2 and 3 (5 micrograms)), polio virus (inactivated) type 1 (40 D antigen units), poliovirus (inactivated type 2 (8 D antigen units), poliovirus (inactivated) type 3 (32 D antigen units), adsorbed on aluminum phosphate (1.5 mg (0.33 mg aluminum)) per 0.5-mL dose.

Immune responses to the diphtheria, tetanus, and pertussis components of REPEVAX (diphtheria toxoid, tetanus toxoid, pertussis toxoid, pertactin, fimbriae types 2 and 3 and filamentous haemagglutinin) were assessed using a multiplexed LUMINEX assay. Immune responses to poliovirus types 1, 2, and 3 were measured in virus neutralization assays. Sera obtained from all subjects in both groups were used in these assays.

For assessment of the immune response to bivalent rLP2086, functional antibodies were analyzed in hSBAs with the 4 primary MnB test strains described. Four primary MnB hSBA test strains (A22, A56, B44, and B24), 2 expressing LP2086 subfamily A and the other 2 expressing LP2086 subfamily B variants were selected. These 4 primary hSBA test strains (from 4 of the 6 fHBP phylogenetic subgroups and representing >90% of disease isolates circulating in the USA and Europe) were used for determination of the primary immunogenicity endpoints in this study. Additionally, the A22, B24, and B44 variants are epidemiologically relevant variants in Europe, while in the US, A22 and B24 are the most prevalent variants found expressed on disease causing MnB strains. The MnB hSBAs were validated prior to testing of samples used for the primary and secondary analyses.

Serum samples from 50% of randomly selected subjects in both groups had hSBA performed with A22 and B24 and the other 50% were tested with A56 and B44. These tests were performed on blood samples collected before Vaccination 1, after Vaccination 2, and after Vaccination 3.

The immunogenicity of REPEVAX is assessed by using prespecified criteria for each antigen defined in the pivotal Phase 3 clinical trials in adolescents that formed the basis of licensure for REPEVAX. The REPEVAX concomitant antigens include diphtheria, tetanus, pertussis toxoid, pertussis filamentous hemagglutinin, pertussis pertactin, pertussis fimbrial agglutinogens type 2+3, poliovirus type 1, poliovirus type 2, poliovirus type 3. The exception is for pertussis fimbrial agglutinogens (FIM) types 2+3, which defined a titer of EU/mL in the assay used for licensure of REPEVAX. In this study the lower limit of quantification (LLOQ) of the pertussis FIM types 2+3 assay was 10.6 EU/mL, which is higher and therefore more stringent than the licensing criteria of REPEVAX.

The LLOQs for the concomitant antigens were 0.037 IU/mL for diphtheria toxoid; 0.05 IU/ml for tetanus toxoid; 0.9 EU/mL for pertussis toxoid; 2.9 EU/mL for pertussis filamentous hemagglutinin, 3.0 EU/mL pertussis pertactin; 10.6 EU/mL pertussis fimbrial agglutinogens type 2+3; 1:8 for poliovirus type 1, poliovirus type 2, poliovirus type 3.

Additional descriptive endpoints for the primary objective were the antibodies to concomitant vaccine antigens measured as geometric mean titer (GMTs) or geometric mean concentrations (GMCs) at postvaccination 1 (Visit 2).

Another endpoint was the proportion of subjects with hSBA titer ≥LLOQ at Postvaccination 3 (Visit 6) for each of the 4 primary MnB test strains.

Concomitant vaccine antigens. The proportion of subjects achieving the prespecified criteria for the concomitant vaccine antigens 1 month after vaccination of diphtheria, tetanus, and pertussis acellular (dTaP)-IPV (REPEVAX) was computed with a 2-sided 95% exact (or Clopper-Pearson confidence limit) for Group 1 and Group 2. The difference (bivalent rLP2086/dTaP-IPV-dTaP-IPV, or Group 1-Group 2) of the proportions was also calculated along with a 2-sided 95% exact CI for the difference. Noninferiority was declared if the lower limit of the 2-sided 95% CI for the difference was greater than −0.10 (−10%) for all of the 9 antigens in the dTaP-IPV vaccine.

hSBAs with Primary Test Strains. For each primary MnB hSBA test strain, the number and proportion of subjects achieving hSBA titers ≥LLOQ, ≥4, ≥8, 1:16, and ≥128 at each blood sampling time point were descriptively summarized along with the exact 2-sided 95% CI (or Clopper-Pearson confidence limit) for the proportion.

Results: Of 749 subjects randomized, 685 (91.5%) included the evaluable immunogenicity population. Immune responses following REPEVAX+rLP2086 or REPEVAX+saline were noninferior for all 9 REPEVAX antigens. Immune responses to the bivalent rLP2086 vaccine were substantial after 2 doses and further enhanced after 3 doses (Table 2). Mild-to-moderate injection site pain was the most common local reaction; headache and fatigue were the most common systemic events. The proportion of subjects reporting an AE within 30 days postvaccination was similar (8.8% and 11.4%, for REPEVAX+rLP2086 and REPEVAX+saline, respectively).

For the concomitant vaccine evaluable immunogenicity population, the proportion of subjects achieving the prespecified level of antibodies to concomitant vaccine antigens (threshold for response) 1 month after the REPEVAX dose was similar between the bivalent rLP2086+REPEVAX group and the REPEVAX alone group for concomitant vaccine antigens: diphtheria toxoid (99.4% in each group), tetanus toxoid (100% in each group), pertussis toxoid (94.7% and 96.0%, respectively), pertussis filamentous hemagglutinin (100% in each group), pertussis pertactin (100% in each group), pertussis fimbrial agglutinogens type 2+3 (97.6% and 98.9%, respectively), poliovirus type 1 (100% in each group), poliovirus type 2 (100% in each group), poliovirus type 3 (100% in each group).

Noninferiority was achieved because the lower bound of the 2-sided 95% CI for the difference in proportion of responders between the bivalent rLP2086+REPEVAX group (Group 1) and the REPEVAX alone group (Group 2), 1 month after the REPEVAX dose was greater than −0.10 (−10%) for the 9 antigens in REPEVAX (i.e., the lowest lower bound of the 95% CI on the proportion difference was −4.7% (pertussis toxoid). Hence, the immune response induced by REPEVAX given with bivalent rLP2086 was noninferior to the immune response induced by REPEVAX alone.

The proportion of subjects with an hSBA titer ≥LLOQ for each of the 4 primary MnB test strains for the Postvaccination 3 evaluable immunogenicity population was assessed. The LLOQ for A22 was an hSBA titer equal to 1:16 while the LLOQ for all the other MnB test stains was an hSBA titer equal to 1:8.

For Group 1, the proportion of subjects with an hSBA titer ≥LLOQ at baseline (before Vaccination 1) was 14.4% for primary MnB strain A22, 18.2% for A56, 12.7% for B24, and 6.2% for B44. For Group 2, the proportion of subjects with an hSBA titer LLOQ at baseline (before Vaccination 1) was 23.0% for primary MnB strain A22, 21.8% for A56, 12.9% for B24, and 6.3% for B44.

Substantial hSBA responses were observed among Group 1 subjects after Dose 2 of bivalent rLP2086, with additional increases observed after 3 doses 1 month after Vaccination 3. For Group 1 (bivalent rLP2086+REPEVAX), the proportion of subjects achieving an hSBA titer ≥LLOQ at 1 month after Vaccination 2 and at 1 month after Vaccination 3 was 81.1% and 95.6% for A22, 97.3% and 100% for A56, 81.0% and 96.8% for B24, and 55.5% and 81.5% for B44. While substantial hSBA responses were achieved after only two bivalent rLP2086 doses, the increase in the proportion of subjects with an hSBA titer ≥LLOQ after 2 doses (1 month after Vaccination 2) compared to 3 doses (1 month after Vaccination 3) exemplifies the enhancement of an immune response after 3 doses. In the control group (Group 2), the proportions of subjects with an hSBA titer ≥LLOQ for each of the 4 primary MnB test strains at 1 month after Vaccination 2 and 1 month after Vaccination 3 were similar to the baseline hSBA results for each MnB test strain (before Vaccination 1).

For the 4 primary MnB test strains, the proportion of subjects in Group 1 exhibiting a defined hSBA titer was greater after 3 doses than after 2 doses. Subjects who achieved an hSBA titer of 1:16 are described, since this titer is a 4-fold increase from a 1:4 titer (a titer of ≥4 is widely recognized as the correlate of protection against IMD). For Group 1, the proportion of subjects with an hSBA titer of 1:16 at 1 month after Vaccination 2 was 81.8% for A22, 97.3% for A56, 68.0% for B24, and 53.4% for B44. One month after Vaccination 3, the proportion of subjects with an hSBA titer of 1:16 was 95.6% for A22, 100% for A56, 87.3% for B24, and 79.5% for B44.

In the control group (Group 2), the proportions of subjects exhibiting defined hSBA titers for each of the 4 primary MnB test strains at 1 month after Vaccination 2 and 1 month after Vaccination 3 were similar to the proportion of subjects with the defined hSBA titer at baseline (before Vaccination 1).

For Group 1, the proportion of subjects with an hSBA titer of 1:16 following 3 doses of bivalent rLP2086 demonstrated that the vaccine elicits a robust immune response when 3 doses of bivalent rLP2086 were administered.

hSBA Geometric Mean Titers (GMTs). In general, the GMTs at baseline were below the hSBA LLOQs for both groups. For Group 1, hSBA GMTs at 1 month after Vaccination 2 were 35.5 for A22, 91.1 for A56, 15.9 for B24, and 14.6 for B44. The hSBA GMTs at 1 month after Vaccination 3 were 63.4 for A22, 151.5 for A56, 28.3 for B24, and 36.5 for B44.

For Group 1, the observed GMTs after 2 doses for subfamily A strains, as well as after 3 doses for subfamily B strains, were indicative of a robust immune response.

Reverse cumulative distribution curves (RCDCs) showing the distribution of hSBA titers for A22, A56, B24, and B44 were assessed. Results from the RCDCs in Group 1 showed that substantial immune responses were observed among Group 1 subjects after Vaccination 2 of bivalent rLP2086, however, the figures also showed the benefit of a third dose of bivalent rLP2086 as greater proportion of subjects achieved higher titers against the 4 MnB test strains. The effect was most pronounced for strain B44.

Conclusions: When given concomitantly with bivalent rLP2086, REPEVAX induced immune responses that were noninferior to those elicited by REPEVAX alone. The bivalent rLP2086 vaccine induced robust bactericidal responses to four diverse MnB test strains, particularly to those representing subfamily B, that were greater after 3 doses than 2 doses. Concomitant administration was generally safe and well tolerated.

TABLE 2 Immune response to 4 heterologous MnB test strains after doses 2 and 3 of bivalent rLP2086 rLP2086 + REPEVAX Saline + REPEVAX Strain [fHBP variant] hSBA ≥ LLOQ hSBA ≥ LLOQ Time point N^(a) hSBA GMT (95% CI)^(c) n^(b) (%) (95% CI)^(d) N^(a) hSBA GMT (95% CI)^(c) n^(b) (%) (95% CI)^(d) PMB80 [A22] Dose 2 154 35.5 (30.27, 41.61) 126 (81.8) (74.8, 87.6) 166  11.2 (10.02, 12.46) 36 (21.7) (15.7, 28.7) Dose 3 158 63.4 (55.29, 72.79) 151 (95.6) (91.1, 98.2) 166 11.0 (9.92, 12.27) 33 (19.9) (14.1, 26.8) PMB2001 [A56] Dose 2 149  91.1 (78.00, 106.51) 145 (97.3) (93.3, 99.3) 151  8.3 (6.76, 10.29) 39 (25.8) (19.1, 33.6) Dose 3 148  151.5 (131.47, 174.59)  148 (100.0)  (97.5, 100.0) 152  8.5 (6.90, 10.54) 40 (26.3) (19.5, 34.1) PMB2948 [B24] Dose 2 153 15.9 (13.55, 18.55) 124 (81.0) (73.9, 86.9) 167 4.8 (4.41, 5.19) 20 (12.0)  (7.5, 17.9) Dose 3 157 28.3 (24.49, 32.66) 152 (96.8) (92.7, 99.0) 170 4.8 (4.41, 5.15) 22 (12.9)  (8.3, 18.9) PMB2707 [B44] Dose 2 146 14.6 (11.6, 18.43)   81 (55.5) (47.0, 63.7) 159 4.7 (4.24, 5.12) 12 (7.5)   (4.0, 12.8) Dose 3 146 36.5 (28.93, 46.18) 119 (81.5) (74.2, 87.4) 159 4.7 (4.29, 5.24) 13 (8.2)   (4.4, 13.6) GMT = geometric mean titer; hSBA = serum bactericidal assay using human complement; LLOQ = lower limit of quantitation (titer 1:16 for PMB80 [A22] and 1:8 for the other MnB test strains); rLP2086 = recombinant lipoprotein 2086. ^(a)Number of subjects with valid hSBA titers for the given strain ^(b)Number of subjects with hSBA titer >LLOQ for given strain at specified time point ^(c)Confidence intervals are back transformations of confidence intervals based on Student t distribution for the mean logarithm of the hSBA titers ^(d)Exact 2-sided confidence intervals based on observed proportion of subjects using the Clopper and Pearson method

Example 5

Immunogenicity of an Investigational Meningococcal Serogroup B Bivalent rLP2086 Vaccine in Healthy Adolescents

Background and Aims: Neisseria meningitidis serogroup B (MnB) causes invasive disease in infants, adolescents, and adults. A conserved, surface-exposed lipoprotein, LP2086 (a factor H binding protein [fHBP]), is a promising MnB vaccine target. Safety and immunogenicity of an investigational bivalent, recombinant vaccine (rLP2086) were studied in healthy adolescents (11-18 years).

Methods: Subjects in this placebo-controlled, single-blind study were randomized to two 3-dose schedules and three 2-dose schedules. Each 120-μg dose contained 2 rLP2086 antigens, 1 from each LP2086 subfamily (A and B). Saline was given when vaccine was not scheduled. Serum bactericidal assays using human complement (hSBA) were performed with 4 MnB test strains (heterologous to vaccine fHBP).

Results: 1713 subjects (mean age, 14.4 y) were randomized. One month after 3 doses of vaccine, hSBA titers to subfamily A and B strains were observed in 95-99% and 86-89% of subjects, respectively; after 2 doses, these numbers ranged from 91-100% and 69-77% of subjects, respectively. Of the 2-dose schedules, 0 and 6 months induced the highest antibody responses (Table 1 of Example 5). hSBA GMTs after 2 doses ranged from 6.2-125.6 and after 3 doses ranged from 25.6-155.6 across the 4 MnB heterologous test strains. Mild-to-moderate injection site pain was the most common local reaction. Fever 38° C. was experienced in 3.3-6.5% and 2.1% of rLP2086 and saline recipients, respectively, after dose 1.

TABLE 1 Proportion of Subjects Achieving hSBA Titer ≥8^(a) for Each Strain 1 Month After Last Dose of Bivalent rLP2086 Group 1 Group 2 Group 3 Group 4 Group 5 (0, 1, 6 mo) (0, 2, 6 mo) (0, 6 mo) (0, 2 mo) (2, 8 mo) Strain n = 354-362 n = 352-359 n = 356-370 n = 234-240 n = 110-113 [fHBP variant] % (95% CI)^(†) % (95% CI)^(†) % (95% CI)^(†) % (95% CI)^(†) % (95% CI)^(†) PMB80 [A22] 91.7^(‡) (88.3, 94.3) 95.0^(‡) (92.1, 97.0) 93.5^(‡) (90.5, 95.8) 90.8 (86.3, 94.1) 91.9 (85.2, 96.2) PMB2001 [A56] 99.4^(‡) (98.0, 99.9) 98.9^(‡) (97.2, 99.7) 98.4^(‡) (96.5, 99.4) 100.0 (98.5, 100.0) 99.1 (95.2, 100.0) PMB2948 [B24] 89.0^(‡) (85.2, 92.0) 88.4^(‡) (84.6, 91.0) 81.1^(‡) (78.6, 85.0) 73.0 (66.9, 78.5) 69.1 (59.6, 77.6) PMB2707 [B44] 88.5^(‡) (84.7, 91.6) 88.1^(‡) (82.0, 89.5) 77.5^(‡) (72.2, 82.3) 70.1 (83.8, 75.9) 73.0 (63.7, 81.0) hSBA = serum bactericidal assay using human complement. ^(a)Lower limit of quantification for all strains = 8. ^(†)Exact 2-sided confidence interval (Copper and Pearson) based upon the observed proportion of subjects. ^(‡)P < 0.001, values <0.0125 are considered significant. P value only apply to Groups 1, 2, and 3.

TABLE 2 hSBA GMTs for Each Strain 1 Month After Last Dose of Bivalent rLP2086 Group 1 Group 2 Group 3 Group 4 Group 5 (0, 1, 6 mo) (0, 2, 6 mo) (0, 6 mo) (0, 2 mo) (2, 8 mo) Strain n = 354-362 n = 352-359 n = 358-370 n = 234-240 n = 110-113 [fHBP variant] GMT^(a) (95% CI)^(†) GMT^(a) (95% CI)^(†) GMT^(a) (95% CI)^(†) GMT^(a) (95% CI)^(†) GMT^(a) (95% CI)^(†) PMB80 [A22] 55.1 (48.87, 62.07) 56.3 (50.91, 62.27) 48.4 (43.45, 53.86) 14.2 (12.08, 16.73) 39.6 (32.31, 49.46) PMB2001 [A56]  152.9 (137.23, 170.47)  158.6 (140.39, 172.38)  126.8 (112.59, 140.17) 26.6 (22.24, 31.68) 111.8 (92.73, 134.90) PMB2948 [B24] 29.1 (26.86, 32.66) 25.6 (23.03, 28.45) 20.6 (18.38, 23.16) 8.0 (7.01, 9.24)  14.7 (12.01, 18.10) PMB2707 [B44] 40.3 (36.16, 46.11) 35.0 (30.63, 39.91) 22.6 (19.80, 26.72) 8.2 (5.52, 7.07)  17.8 (14.12, 22.42) GMT = geometric mean titer; hSBA = serum bactericidal assay using human complement. ^(a)GMTs were calculated using as subjects with valid and determinate hSBA titers at the given time point. ^(†)CIs are back transformations of confidence levels based on the Student t distribution for the mean logarithm of the hSBA titers. Conclusions: rLP2086 was well tolerated. All dosing regimens yielded robust bactericidal responses that were most pronounced after 3 doses.

Table 1 of Example 5 is the same as Table 1 of Example 1, described above. Table 2 summarizes the hSBA GMTs and the corresponding Cls by study time for the evaluable immunogenicity population. GMTs increased from baseline (before Injection 1) and continued to increase with each subsequent dose of bivalent rLP2086.

For the 4 primary MnB strains, the GMTs were greater after 3 doses of bivalent rLP2086 (Groups 1 and 2) than after 2 doses (Groups 3, 4, and 5). The GMTs were similar between the two 3-dose groups, and they were similar among the three 2-dose groups.

Before injection 1 (baseline), hSBA GMTs for Groups 1, 2, 3, 4, and 5 were as follows: 7.1, 6.3, 6.4, 6.4, and 6.8 for A22, respectively; 6.8, 6.1, 6.7, 6.3, and 6.2 for A56, respectively; 5.3, 5.1, 5.0, 4.9, and 5.1 for B24, respectively; and 4.4, 4.5, 4.5, 4.6, and 4.4 for B44, respectively.

For Group 1 (0-, 1-, and 6-month), there was a substantial increase in GMTs noted 1 month after Dose 2 for all 4 primary MnB strains (24.4, 77.3, 13.8, and 13.1 for A22, A56, B24, and B44, respectively). The GMTs further increased after 3 doses of bivalent rLP2086 for Group 1 subjects for the 4 primary MnB test strains; 55.1 (A22); 152.96 (A56); 29.1 (B24); and 40.3 (B44).

For Group 2, similar increases in GMTs were noted after 2 and 3 doses of bivalent rLP2086. GMTs for Group 2 subjects after 2 doses of bivalent rLP2086 were 32.9 for A22; 94.6 for A56; 14.9 for B24; and 15.5 for B44. After 3 doses, the GMTs increased to 56.3 for A22; 155.6 for A56; 25.6 for B24; and 35.0 for B44.

For Groups 1 and 2, the observed GMTs after 2 doses for subfamily A strains, as well as after 3 doses for subfamily B strains, are indicative of a robust immune response.

For Group 3, small increases in GMTs were noted after 1 dose of bivalent rLP2086 as follows: 12.0 for A22; 18.5 for A56; 9.2 for B24; and 5.7 for B44. After 2 doses GMTs increased to 48.4 for A22; 125.6 for A56; 20.6 for B24; and 22.5 for B44.

For Group 4, GMTs were 13.3 for A22; 17.7 for A56; 9.8 for B24; and 5.9 for B44 after 1 dose of bivalent rLP2086. After 2 doses of bivalent rLP2086, GMTs were 37.1 for A22; 104.9 for A56; 17.7 for B24; and 19.1 for B44.

For Group 5, GMTs after 1 dose of bivalent rLP2086 were 16.0 for A22; 26.8 for A56; 12.6 for B24; and 6.8 for B44. After 2 doses of bivalent rLP2086, the GMTs increased to 39.6 for A22; 111.8 for A56; 14.7 for B24; and 17.8 for B44.

Taken together, for Groups 3, 4, and 5, the observed GMTs are indicative of an immune response for subfamily A and B strains after 2 doses of bivalent rLP2086.

In summary, 3 doses of bivalent rLP2086 provided a robust and the broadest immune response based on the hSBA titers for the 4 primary MnB test strains. In comparison to 2 doses, a higher proportion of subjects receiving 3 doses of bivalent rLP2086 achieved an hSBA titer ≥8 to the 4 primary MnB test strains.

The results following the 0-, 1-, and 6-month dosing schedule (Group 1) were similar to the results following the 0-, 2-, and 6-month dosing schedule (Group 2). For Groups 1 and 2, the post-Dose 3 GMT values achieved were higher than the post-Dose 2 GMT values. For Groups 1 and 2, the post-Dose 2 GMT values ranged from 24.4 to 94.6 for subfamily A strains and from 13.1 to 15.5 for subfamily B strains. The post-Dose 3 GMT values ranged from 55.1 to 155.6 for subfamily A strains and from 25.6 to 40.3 for subfamily B strains. For Groups 1 and 2, a higher proportion of subjects achieved an hSBA titer ≥8 to the 4 primary MnB test strains following 3 doses of bivalent rLP2086 when compared to the proportion of subjects achieving an hSBA titer ≥8 to the 4 primary MnB test strains after 2 doses of bivalent rLP2086.

Subjects who achieved an hSBA titer of 1:16 were also assessed. For Group 1, the percentage of subjects who achieved an hSBA titer of 1:16 one month after 2 doses of bivalent rLP2086 was 73.5% for A22; 96.3 for A56; 57.6 for B24; and 47.2% for B44. Following 3 doses of bivalent rLP2086, the percentage of subjects in Group 1 who achieved an hSBA titer of ≥16 was 91.4% for A22; 99.2% for A56; 82.8% for B24; and 84.8% for B44.

For Group 2, the percentage of subjects who achieved an hSBA titer of ≥16 one month after 2 doses of bivalent rLP2086 was 88.1% for A22; 97.9% for A56; 63.5% for B24; and 58.6% for B44. Following 3 doses of bivalent rLP2086, the percentage of subjects in Group 2 who achieved an hSBA titer of ≥16 was 95.0% for A22; 98.9% for A56; 83.6% for B24; and 83.8% for B44.

For Groups 1 and 2, the percentage of subjects achieving an hSBA titer of ≥16 following 3 doses of bivalent rLP2086 demonstrated that the vaccine elicits a robust immune response.

For Group 3, the percentage of subjects who achieved an hSBA titer of ≥16 after 2 doses of bivalent rLP2086 was 93.2% for A22; 98.4% for A56; 73.8% for B24; and 70.8% for B44.

For Group 4, the percentage of subjects who achieved an hSBA titer of ≥16 one month after 2 doses of bivalent rLP2086 was 90.8% for A22; 99.2% for A56; 67.1% for B24; and 64.5% for B44.

For Group 5, the percentage of subjects who achieved an hSBA titer of ≥16 after 2 doses of bivalent rLP2086 was 91.0% for A22; 99.1% for A56; 64.5% for B24; and 66.7% for B44.

For Groups 3, 4, and 5, the percentage of subjects achieving an hSBA titer of ≥16 demonstrated that the vaccine elicits a robust immune response to subfamily A strains following only 2 doses. However, 3 doses increases the robustness of response to subfamily B strains.

The percentage of subjects achieving an hSBA titer of ≥16 after 3 doses of bivalent rLP2086 shows that the vaccine elicits a robust and broad immune response to MnB strains expressing LP2086 variants that are different from the vaccine components.

Reverse cumulative distribution curves (RCDCs) showing the distribution of hSBA titers by study times were also assessed for the evaluable immunogenicity populations for each strain by group. The RCDCs show robust immune responses after 2 doses of bivalent rLP2086 subfamily A strains. Following the third dose of bivalent rLP2086, the area under the response curves increases for all 4 primary MnB test strains, thereby demonstrating the enhancement of the immune response after 3 doses of bivalent rLP2086.

The results from the primary and secondary immunogenicity endpoint analyses show that the vaccine can generate antibodies with significant hSBA activity against heterologous subfamily A and subfamily B variants of MnB. While the proportion of subjects achieving an hSBA titer ≥8 was higher after 2 or 3 doses of bivalent rLP2086, a large proportion of subjects achieved an hSBA titer ≥8 one month after 1 dose of bivalent rLP2086. See Group 5 for example.

For the 4 primary MnB test strains, the GMTs were greater after 3 doses of bivalent rLP2086 (Groups 1 and 2) than after 2 doses (Groups 3, 4, and 5). The GMTs were similar in the two 3-dose groups. The GMTs were also similar among the three 2-dose groups. These data also demonstrate robust hSBA responses after 3 doses of bivalent rLP2086 based on the percentages of subjects achieving an hSBA titer ≥16.

These data demonstrate that the final formulation of bivalent rLP2086 generates a robust immune response and is safe and well tolerated when given in 2 or 3 doses. Even 1 dose of bivalent rLP2086 provides a substantial immune response above baseline and is also safe and well tolerated. Overall, there was no clinically meaningful difference in the safety profile after 2 or 3 doses of bivalent rLP2086.

Example 6

Safety, Tolerability, and Immunogenicity of a Meningococcal Serogroup B Bivalent rLP2086 Vaccine in Healthy Adolescents Aged 11 to 18 Years in Three Phase 2, Randomized, Controlled Studies

Background: Neisseria meningitidis serogroup B (MnB) is a major cause of invasive meningococcal disease in adolescents. A conserved, surface-exposed lipoprotein, LP2086 (factor H binding protein [fHBP]), is a promising vaccine target to protect against invasive disease caused by MnB. Safety, tolerability, and immunogenicity of an investigational bivalent, recombinant MnB vaccine (including SEQ ID NO: 1 and SEQ ID NO: 2, 2.8 molar ratio polysorbate-80, 0.5 mg/ml aluminum, 10 mM histidine, and 150 mM sodium chloride, herein referred to throughout the Examples as “bivalent rLP2086”) were examined in three phase 2, randomized, controlled studies in healthy adolescents 11-18 years of age.

Methods: Study 1012 examined 5 vaccine regimens of bivalent rLP2086, whereas studies 1010 and 1011 evaluated a 3-dose schedule of bivalent rLP2086 vaccine given concomitantly with the TdaP-IPV and HPV-vaccines, respectively. Each dose of bivalent rLP2086 contained 60 μg of the rLP2086 subfamily A variant A05 and 60 μg of the rLP2086 subfamily B variant B01. To examine immunogenicity of bivalent rLP2086 in each of the three studies, serum bactericidal assays using human complement (hSBA) were performed with 4 MnB test strains expressing the heterologous fHBP variants A22, A56, B24 and B44, which were selected to represent relevant diversity of fHBP variability, as well as to provide a perspective on the breadth of the vaccine-elicited immune response against strain expressing epidemiologically prevalent fHBP variants. Adverse events and solicited local and systemic reactions were assessed.

Results: 82-100% of subjects in all 3 studies achieved hSBA titers above the lower limit of quantification (LLOQ) for each of the 4 MnB test strains 1 month after dose 3 (Table). Across the three studies, the majority of systemic events and local reactions were mild to moderate in severity; adverse events were generally not serious or related to the study vaccine.

Conclusions: Serum bactericidal antibody titers above 1:4 protect against invasive meningococcal disease. The demonstration of hSBA titers ≥LLOQ to 4 MnB test strains, each heterologous to vaccine antigen, in each of these adolescent phase 2 studies, suggest that the bivalent rLP2086 vaccine provided a functional antibody response that may be broadly active against diverse MnB disease-associated strains. Vaccinations with the bivalent rLP2086 were generally well tolerated.

TABLE Proportion of Subjects Achieving an hSBA Titer ≥LLOQ for Each fHBP Variant Expressed by Each Test Strain 1 Month After the Last Dose of the Bivalent rLP2086 Vaccine fHBP variant expressed % of Subjects by hSBA test strain A22 A56 B24 B44 Study 1012 (dosing regimen) Group 1 (0, 1, 6 mo); n = 354-360 91.4 99.4 89.0 88.5 Group 2 (0, 2, 6 mo); n = 352-359 95.0 98.9 88.4 86.1 Group 3 (0, 6 mo); n = 356-370 93.2 98.4 81.1 77.5 Group 4 (0, 2 mo); n = 234-240 90.8 100.0 73.0 70.1 Group 5 (0, 4 mo); n = 110-113 91.0 99.1 69.1 73.0 Study 1010 (dosing regimen: 0, 2, 6 mo) rLP2086 + TdaP-IPV Vaccine; n = 146-158 95.6 100.0 96.8 81.5 Study 1011 (dosing regimen: 0, 2, 6 mo) rLP2086 + HPV Vaccine; n = 833-849 94.0 98.9 90.5 82.7 rLP2086 + Saline; n = 847-848 96.3 99.4 92.6 85.7 LLOQ = lower limit of quantification; fHBP = factor H binding protein; hSBA = serum bactericidal assay using human complement; TdaP-IPV Vaccine = Tetanus, Diphtheria, Pertussis, Polio Vaccine. LLOQ = the lowest amount of an analyte in a sample that can be quantitatively determined. hSBA titer ≤ ≥ 1:4 are a correlate of protection for invasive meningococcal disease. hSBA titers ≥LLOQ are above the minimal correlate. LLOQ was 1:16 for A22; and 1:8 for A56, B24, and B44.

Example 7

Immunogenicity of a Meningococcal Serogroup B Bivalent rLP2086 Vaccine in Healthy Adolescents Aged 11 to 18 When Administered Concomitantly With Human Papillomavirus Vaccine

This Phase 2, randomized, observer-blind, controlled study evaluated the immunogenicity of bivalent rLP2086 with or without coadministration with GARDASIL®, which is a quadrivalent vaccine against human papillomavirus (HPV4) (as also described in U.S. Pat. No. 5,820,870), in healthy adolescents ≥11 to <18 years of age. GARDASIL contains recombinant antigens of HPV type 6, 11, 16, and 18 (i.e., HPV-6, HPV-11, HPV-16, and HPV-18) L1 protein. An endpoint was the hSBA GMTs for each of the 4 primary MnB test strains at each applicable blood sampling time point.

Methods: Subjects received bivalent rLP2086 (including SEQ ID NO: 1 and SEQ ID NO: 2, 2.8 molar ratio polysorbate-80, 0.5 mg/ml aluminum, 10 mM histidine, and 150 mM sodium chloride)+HPV4 (Group 1), bivalent rLP2086+saline (Group 2), or HPV4+saline (Group 3) at months 0, 2, and 6. Sera from subjects in Groups 1 and 2 before vaccination 1, and 1 month after vaccinations 2 and 3, were tested by serum bactericidal assay using human complement (hSBA) using 4 MnB test strains, each expressing an fHBP (A22, A56, B44, and B24) that is heterologous to the vaccine components and represents the breadth of fHBP diversity, as well as epidemiological prevalence. Endpoints assessed included the proportion of subjects with hSBA titers the lower limit of quantitation (LLOQ; 1:16 [A22] or 1:8 [A56, B44, B24]) and hSBA geometric mean titers (GMTs).

To demonstrate noninferiority of administrating GARDASIL plus bivalent rLP2086 compared to GARDASIL alone, immunogenicity assessments were performed with 2 hSBAs, using 1 primary test strain representing subfamily A variants (A22) and 1 primary test strain representing subfamily B variants (B24). However, all 4 primary MnB test strains were used for determination of additional bivalent rLP2086 immunogenicity/efficacy exploratory endpoints.

For assessment of the immune response to bivalent rLP2086, functional antibodies were analyzed in hSBAs with meningococcal serogroup B strains randomly selected from Pfizer's representative MnB SBA strain pool, as described in Example 2. The hSBAs measured the functional antibodies in human sera that in a complement-dependent manner kill the target meningococcal strain.

Results: 814 and 812 subjects included the evaluable immunogenicity population for Groups 1 and 2, respectively. Compared with before vaccination 1, the proportion of subjects with hSBA titers ≥LLOQ against all 4 test strains was higher after vaccinations 2 (55%-99%) and 3 (83%-99%; FIG. 1). Table A of Example 7 presents the hSBA GMTs for each of the 4 primary MnB strains and the corresponding Cls by sampling time point for the evaluable immunogenicity population. The GMTs at baseline were below the hSBA LLOQs for both groups. GMTs ranged from 11.1-70.6 and 11.9-76.3 after vaccination 1, and 25.8-117.2 and 28.0-128.2 after vaccination 2 in Groups 1 and 2, respectively (Table A below).

For the evaluable immunogenicity population, the hSBA GMTs to the 2 primary MnB strains at 1 month after the Vaccination 3 bivalent rLP2086 dose for Group 1 and Group 2 were as follows: 53.3 and 57.8, respectively for A22 and 25.8 and 28.0, respectively for B24.

For Group 2 (bivalent rLP2086+saline), hSBA GMTs at 1 month after Vaccination 2 were 33.7 for A22, 76.3 for A56, 16.3 for B24, and 11.9 for B44. The hSBA GMTs at 1 month after Vaccination 3 were 57.8 for A22, 128.2 for A56, 28.0 for B24, and 31.9 for B44.

For Group 1 (bivalent rLP2086+GARDASIL), hSBA GMTs at 1 month after Vaccination 2 were 31.9 for A22, 70.6 for A56, 15.0 for B24, and 11.1 for B44. The hSBA GMTs at 1 month after Vaccination 3 were 53.3 for A22, 117.2 for A56, 25.8 for B24, and 27.2 for B44.

Reverse cumulative distribution curves (RCDCs) showing the distribution of hSBA titers for A22, A56, B24, and B44 were assessed for Group 1 and Group 2 at all sampling time points for the evaluable immunogenicity population. The RCDCs showed that the majority of subjects responded after Vaccination 2 and had an additional increase in titer for the 4 primary MnB test strains after Vaccination 3. Immune responses to the antigens were similar for Groups 1 and 2.

Conclusions: Bivalent rLP2086 can be administered with HPV4 without affecting the bactericidal response assessed by hSBA seroresponse or GMTs. Since hSBA titers 1:4 correlate with protection against meningococcal disease, these data indicate the potential for protection of adolescents against a broad range of MnB strains following administration of the bivalent rLP2086 in the setting of concomitant administration of HPV vaccine.

TABLE A hSBA GMTs - Evaluable Immunogenicity Population Group 1 Group 2 Strain [Variant] rLP2086 + HPV4 rLP2086 + Saline Sampling Time Point n^(a) GMT^(b) (95% CI)^(c) n^(a) GMT^(b) (95% CI)^(c) PMB80 [A22] Before 794 9.6 (9.3, 10.0) 799 9.9 (9.5, 10.3) Vaccination 1 1 Month After 794 31.9 (29.96, 33.94) 801 33.7 (31.69, 35.85) Vaccination 2 1 Month After 803 53.3 (50.22, 56.66) 801 57.8 (54.44, 61.44) Vaccination 3 PMB2001 [A56] Before 757 5.0 (4.78, 5.32) 740 5.0 (4.75, 5.28) Vaccination 1 1 Month After 790 70.6 (66.17, 75.34) 795 76.3 (71.93, 80.99) Vaccination 2 1 Month After 796 117.2 (110.14, 124.76) 802 128.2 (120.65, 136.27) Vaccination 3 PMB2948 [B24] Before 801 4.3 (4.23, 4.46) 793 4.5 (4.35, 4.65) Vaccination 1 1 Month After 770 15.0 (13.88, 16.15) 770 16.3 (15.15, 17.62) Vaccination 2 1 Month After 788 25.8 (24.14, 27.56) 793 28.0 (26.24, 29.87) Vaccination 3 PMB2702 [B44] Before 806 4.1 (4.04, 4.15) 805 4.2 (4.10, 4.31) Vaccination 1 1 Month After 783 11.1 (10.21, 12.01) 776 11.9 (10.94, 12.96) Vaccination 2 1 Month After 799 27.2 (24.99, 29.68) 795 31.9 (29.25, 34.82) Vaccination 3 GMT = geometric mean titer; HPV4 = quadrivalent human papillomavirus vaccine; hSBA = serum bactericidal assay using human complement. ^(a)n = number of subjects with valid and determinate hSBA titers for the given strain. ^(b)Geometric mean titers were calculated using all subjects with valid and determinate hSBA titers at the given time point. ^(c)Confidence intervals are back transformations of confidence intervals based on the Student t distribution for the man logarithm of the hSBA titers.

Example 8

Immunogenicity of Human Papilloma Vaccine Coadministered with a Bivalent rLP2086 Vaccine Against Meningococcal Serogroup B in Healthy Adolescents

Background: This Phase 2, randomized study evaluated coadministration of a quadrivalent vaccine against human papillomavirus (HPV4), with bivalent rLP2086, an investigational vaccine against invasive disease caused by Neisseria meningitidis serogroup B (MnB), in healthy adolescents ≥11 to <18 years of age.

Methods: Subjects received HPV4+bivalent rLP2086 (Group 1), bivalent rLP2086+saline (Group 2), or saline+HPV4 (Group 3) at months 0, 2, and 6. Sera were collected at baseline and after doses 2 and 3 in all groups. Immune responses to HPV4 antigens (HPV-6, 11, 16, and 18) were determined by competitive LUMINEX immunoassays (cLIAs). Bivalent rLP2086 immunogenicity was measured by serum bactericidal assay using human complement (hSBA) with 2 MnB test strains expressing vaccine-heterologous fHBP variants (A22 and B24). Immunogenicity endpoints, all after dose 3, included: geometric mean titers (GMTs) against HPV antigens in Groups 1 and 3; hSBA GMTs for strains expressing variants A22 and B24 in Groups 1 and 2; and seroconversion rate for HPV antigens in baseline seronegative subjects in Groups 1 and 3. Safety of bivalent rLP2086 was also assessed after concomitant administration with HPV4 or saline.

Assessments of the immune response to GARDASIL (HPV type 6, 11, 16, and 18 L1 protein) were performed using cLIAs based on a fluorescently labeled microsphere-based platform (LUMINEX). Sera obtained from all subjects in Groups 1 and 3 prior to the first vaccination with GARDASIL (Visit 1) and 1 month after the third vaccination with GARDASIL (Visit 5) were used in these assays.

The comparison of the GMTs to the 4 HPV antigens for Group 1 and Group 3, with their corresponding GMT ratios (GMRs) of Group 1 to Group 3 and the 2-sided 95% Cls of the ratios is presented in Table A below of the present Example. The criterion for the noninferiority margin was 1.5-fold, which corresponds to a value of 0.67 for the lower limit of the 2-sided 95% CI of the GMR. The 1.5-fold criterion of 0.67 was met for all the MnB test strains and the HPV antigens except for HPV-18, which had a lower bound 95% confidence interval (CI) of 0.62. In a separate analysis, 99% of subjects seroconverted to all 4 HPV antigens in both the Saline+HPV4 and rLP2086+HPV4 groups.

Another objective of this study was to describe the immune response induced by bivalent rLP2086+GARDASIL (Group 1) and by saline+GARDASIL (Group 3), as measured by seroconversion in the HPV immunogenicity assays after the Vaccination 3 dose of GARDASIL (Visit 5) in both groups.

The seroconversion rate for each of the 4 HPV antigens, 1 month after the last dose of GARDASIL for subjects who were HPV-seronegative at baseline in Group 1 and Group 3, was calculated as the proportion of subjects with anti-HPV serum cLIA levels 20 mMU/ml for HPV-6, mMU/ml for HPV-11, 20 mMU/ml for HPV-16, and 24 mMU/ml for HPV-18.

The number and proportion of baseline HPV-seronegative subjects achieving the prespecified criteria for seroconversion for the 4 HPV antigens with the corresponding 95% Cls in each group, the percent differences (Group 1-Group 3) in the proportion, and the 95% Cls of the differences are presented in Table B of Example 8 for the baseline HPV-seronegative evaluable immunogenicity population.

Results: The prespecified noninferiority criteria set at 1.5-fold (0.67 lower limit of 95% CI for GMRs) were met for 3 of 4 HPV antigens (not HPV-18) and both MnB test strains (Table A). Seroconversion rates in Groups 1 and 3 were 99% for all HPV antigens (Table B). Greater local reactogenicity occurred after rLP2086 compared with saline but did not increase with later doses; injection site pain was the most common local reaction. Systemic events in all 3 groups were generally mild and moderate in severity.

For the evaluable immunogenicity population, the GMTs of antibodies to the 4 HPV antigens at 1 month after the GARDASIL dose at Vaccination 3 for Group 1 and Group 3 were as follows: 451.8 and 550.3, respectively (HPV-6); 892.9 and 1084.3, respectively (HPV-11); 3695.4 and 4763.4, respectively (HPV-16); and 744.0 and 1047.4, respectively (HPV-18). The GMRs of Group 1 to Group 3 at 1 month after the GARDASIL dose at Vaccination 3 were 0.82 for HPV-6 (95% Cl: 0.72, 0.94), 0.82 for HPV-11 (95% Cl: 0.74, 0.91), 0.78 for HPV-16 (95% Cl: 0.68, 0.88), and 0.71 for HPV-18 (95% Cl: 0.62, 0.81). Therefore, the lower limits of the 2-sided 95% Cls for anti-HPV GMRs for Group 1 compared with Group 3 were 0.72 for HPV-6, 0.74 for HPV-11, 0.68 for HPV-16, and 0.62 for HPV-18. The 1.5-fold criterion of 0.67 (the lower limit of the 2-sided 95% CI of the GMR) was met for all HPV antigens except for HPV-18, which had a lower bound of the 95% CI of 0.62.

The GMRs of the bivalent rLP2086+GARDASIL group to the bivalent rLP2086+saline group at 1 month after the Vaccination 3 bivalent rLP2086 dose were 0.92 for A22 (95% Cl: 0.85, 1.00), and 0.92 for B24 (95% Cl: 0.84, 1.01). The lower limits of the 2-sided 95% Cls for the hSBA GMRs for Group 1 compared with Group 2 were 0.85 for A22 and 0.84 for B24, which are both greater than 0.67 and therefore met the noninferiority margin of 1.5-fold.

The data from bivalent rLP2086+GARDASIL (Group 1) administration were compared to data from the bivalent rLP2086+saline (Group 2) administration by analyzing the hSBA titer 4-fold response rates for 2 primary MnB strains (A22 and B24) at 1 month after Vaccination 3 The proportions of subjects achieving ≥4-fold rise in hSBA titer from baseline to 1 month after Vaccination 3 for the 2 primary MnB strains were measured for both Group 1 subjects who received bivalent rLP2086+GARDASIL and Group 2 subjects who received bivalent rLP2086+saline. Of the subjects in Group 1, 85.3% exhibited ≥4-fold rise in hSBA titers against B24. Of the subjects in Group 2, 86.4% exhibited ≥4-fold rise in hSBA titers against A22, and 84.8% exhibited ≥4-fold rise in hSBA titers against B24.

The difference in the proportion of responders between Group 1 and Group 2 at 1 month after Vaccination 3 was −1.1% for A22 (95% Cl: −4.6, 2.3) and −1.4% for B24 (95% Cl: −5.1, 2.3). The differences of 4-fold response rates were all near a value of 1%, with the lower bounds of the 95% Cl of the proportion difference being −4.6% A22 and −5.1% B24.

The noninferiority criteria of bivalent rLP2086+GARDASIL compared to saline+GARDASIL or compared to bivalent rLP2086+saline required that the lower limit of the 2-sided 95% Cls for the GMRs for antibodies to HPV for all 4 HPV antigens (HPV-6, HPV-11, HPV-16, and HPV-18) and for hSBA titers using 2 primary MnB test strains (A22 and B24) 1 month after Vaccination 3 be greater than 0.67. This prespecified criterion was met for both MnB test strains and at least 3 of the 4 HPV antigens. For HPV-18, the lower limit of the 2-sided Cls for the GMR was slightly below the prespecified threshold of 0.67, at 0.62.

The 4-fold rise responses to 2 primary MnB test strains (A22 and B24) were similar (ranged from 83.4% to 86.4%) between the group that received bivalent rLP2086+GARDASIL and the group that received bivalent rLP2086+saline.

The proportions of subjects in Groups 1 and 2 with prevaccination (i.e., before Vaccination 1) hSBA titers of ≥4 were 15.2% and 18.8%, respectively, for strain A22; 10.4% and 10.5%, respectively, for strain A56; 6.1% and 8.4%, respectively, for strain B24; and 1.7% and 3.2%, respectively for strain B44. In addition, the proportions of subjects in Groups 2 and 1 with prevaccination hSBA titers of ≥16 were 13.7% and 16.4%, respectively for strain A22; 9.0% and 9.1%, respectively, for strain A56; 4.1% and 5.4%, respectively, for strain B24; and 1.2% and 2.1%, respectively, for strain B44.

In Group 2 (bivalent rLP2086+saline), the proportion of subjects with an hSBA titer ≥4 at 1 month after Vaccination 2 was 86.3% for A22, 98.7% for A56, 77.1% for B24, and 60.1% for B44. One month after Vaccination 3, the proportion of subjects with an hSBA titer of ≥4 was 96.4% for A22, 99.4% for A56, 92.8% for B24, and 86.5% for B44. In Group 1 (bivalent rLP2086+GARDASIL), the proportion of subjects with an hSBA titer of ≥4 at 1 month after Vaccination 2 was 83.8% for A22, 97.8% for A56, 71.9% for B24, and 57.7% for B44. One month after Vaccination 3, the proportion of subjects with an hSBA titer of ≥4 was 94.3% for A22, 99.1% for A56, 91.1% for B24, and 84.4% for B44.

In Group 2 (bivalent rLP2086+saline), the proportion of subjects with an hSBA titer ≥16 at 1 month after Vaccination 2 was 85.8% for A22, 98.4% for A56, 68.8% for B24, and 49.9% for B44. One month after Vaccination 3, the proportion of subjects with an hSBA titer of ≥16 was 96.3% for A22, 99.4% for A56, 89.2% for B24, and 82.4% for B44. In Group 1 (bivalent rLP2086+GARDASIL), the proportion of subjects with an hSBA titer of ≥16 at 1 month after Vaccination 2 was 83.0% for A22, 97.2% for A56, 65.2% for B24, and 46.4% for B44. One month after Vaccination 3, the proportion of subjects with an hSBA titer of ≥16 was 94.0% for A22, 98.9% for A56, 86.3% for B24, and 78.0% for B44.

For both Group 1 and Group 2, a high proportion of subjects achieved an hSBA titer of ≥16 or greater following 2 or 3 doses of bivalent rLP2086, while most of the subjects had no measureable hSBA titer to any of the primary MnB test strains at prevaccination Visit 1.

For the baseline HPV-seronegative evaluable immunogenicity population, the proportion of subjects achieving the prespecified criteria for HPV seroconversion for the HPV antigens at 1 month after the GARDASIL dose at Vaccination 3 for the bivalent rLP2086+GARDASIL group (Group 1) and the saline+GARDASIL group (Group 3) were as follows: HPV-6 (99.4% and 99.3%, respectively), HPV-11 (99.6% and 99.5%, respectively), HPV-16 (99.6% and 99.5%, respectively), and HPV-18 (99.5% and 99.0%, respectively).

The difference in proportion of responders between the bivalent rLP2086+GARDASIL group (Group 1) and the saline+GARDASIL group (Group 3) at 1 month after the GARDASIL dose was 0.1% for HPV-6 (95% Cl; −0.9, 1.5), 0.1% for HPV-11 (95% Cl: −0.7, 1.3), 0.1% for HPV-16 (95% Cl; −0.7, 1.3), and 0.5% for HPV-18 (95% Cl; −0.6, 1.9).

For the bivalent rLP2086+GARDASIL group (Group 1) and the saline+GARDASIL group (Group 3), the seroconversion rate differences were within 0.1% and 0.5% across all 4 HPV antigens and the seroconversion rates were very similar across groups, with greater than 99% of subjects seroconverting for all 4 HPV antigens.

As an additional evaluation, bivalent rLP2086+GARDASIL (Group 1) was compared to bivalent rLP2086+saline (Group 2), by analyzing the hSBA titer 4-fold response rates for 2 primary MnB strains (A22 and B24) at 1 month after Vaccination 3. The proportions of subjects achieving an hSBA titer fold rise ≥4 from baseline to 1 month after Vaccination 3 for the 2 primary MnB strains are as follows: Of the subjects in Group 1, 85.3% exhibited ≥4-fold rise in hSBA titers against test strain A22, and 83.4% exhibited ≥4-fold rise in hSBA titers against test strain B24. Of the subjects in Group 2, 86.4% exhibited ≥4-fold rise in hSBA titers against test strain A22 and 84.8% exhibited ≥4-fold rise in hSBA titers against test strain B24.

The difference in the proportion of responders between Group 1 and Group 2 at 1 month after Vaccination 3 was −1.1% for A22 (95% Cl: −4.6, 2.3) and −1.4% for B24 (95% Cl: −5.1, 2.3). The differences of 4-fold response rate were all near a value of 1%, with the lower bounds of the 95% CI of the proportion difference being −4.6% (A22) and −5.1% (B24).

Immune responses to bivalent rLP2086. Another objective of this study was to describe the immune response as measured by hSBA performed with 4 primary MnB test strains, 2 expressing LP2086 subfamily A proteins (A22 and A56) and 2 expressing LP2086 subfamily B proteins (B24 and B44), measured 1 month after the second visit (Visit 3) and the third (Visit 5) vaccinations with bivalent rLP2086.

One of the endpoints for this objective was the proportion of subjects with hSBA titers ≥LLOQ at 1 month after Vaccination 2 (Visit 3) and at 1 month after Vaccination 3 (Visit 5) for each of the 4 primary MnB test strains. The proportion of subjects with hSBA titer ≥LLOQ for each of the 4 primary MnB test strains for the evaluable immunogenicity population was assessed. The LLOQ for A22 was an hSBA titer equal to 1:16, while the LLOQ for all the other MnB test strains was an hSBA titer equal to 1:8.

For Group 2 (bivalent rLP2086+saline), the proportion of subjects with an hSBA titer ∛LLOQ at baseline (before Vaccination 1) was 16.4% for A22, 9.3% for A56, 6.9% for B24, and 2.5% for B44. For Group 2, the proportions of subjects achieving an hSBA titer ≥LLOQ at 1 month after Vaccination 2 and at 1 month after Vaccination 3 were 85.8% and 96.3%, respectively, for A22; 98.5% and 99.4%, respectively, for A56; 74.2% and 92.6%, respectively for B24; and 57.1% and 85.7%, respectively, for B44.

For Group 1 (bivalent rLP2086+GARDASIL), the proportion of subjects with an hSBA titer ≥LLOQ at baseline (before Vaccination 1) was 13.7% for A22, 9.2% for A56, 5.1% for B24, and 1.4% for B44. For Group 1, the proportions of subjects achieving an hSBA titer ≥LLOQ at 1 month after Vaccination 2 and at 1 month after Vaccination 3 were 83.0% and 94.0%, respectively, for A22; 97.5% and 98.9%, respectively, for A56; 70.6% and 90.5%, respectively for B24; and 54.5% and 82.7%, respectively, for B44.

Substantial hSBA responses to the 4 primary MnB test strains were observed among both Group 1 and Group 2 subjects at 1 month after Vaccination 2, with additional increases observed at 1 month after Vaccination 3.

The proportion of subjects achieving an hSBA titer fold rise ≥4 for each of the 4 primary MnB test strains and the proportions of subjects achieving the composite response for the evaluable immunogenicity population were assessed. The proportions of subjects with an observed hSBA titer ≥LLOQ for all 4 MnB strains combined at baseline (before Vaccination 1) were similar between Group 1 (0.3%) and Group 2 (0.7%).

For Group 2 (bivalent rLP2086+saline), the proportion of subjects achieving an hSBA titer fold rise ≥4 from baseline to 1 month after Vaccination 3 was 86.4% for A22, 95.3% for A56, 84.8% for B24, and 80.7% for B44, and 83.9% of subjects achieved a composite hSBA response (hSBA LLOQ for all 4 primary strains combined). At 1 month after Vaccination 2, the proportion of subjects achieving an hSBA titer fold rise from baseline was 74.2% for A22, 92.6% for A56, 63.4% for B24, and 47.4% for B44, and 51.9% of subjects achieved a composite hSBA response.

For Group 1 (bivalent rLP2086+saline), the proportion of subjects achieving an hSBA titer fold rise ≥4 from baseline to 1 month after Vaccination 3 was 86.4% for A22, 95.3% for A56, 84.8% for B24, and 80.7% for B44, and 83.9% of subjects achieved a composite hSBA response (hSBA LLOQ for all 4 primary strains combined). At 1 month after Vaccination 2, the proportion of subjects achieving an hSBA titer fold rise from baseline was 74.2% for A22, 92.6% for A56, 63.4% for B24, and 47.4% for B44, and 51.9% of subjects achieved a composite hSBA response.

Additional hSBA fold response. Other endpoints were the proportion of subjects achieving at least 2-fold and 3-fold hSBA titer increases from baseline to each postvaccination blood sampling visit for each of the 4 primary MnB strains. Note that the LLOQ for A22 was an hSBA titer equal to 1:16, while the LLOQ for all the other MnB test strains was an hSBA titer equal to 1:8.

The proportion of subjects achieving a 2-fold rise in hSBA titer from baseline to 1 month after Vaccination 2 for Group 1 and Group 2 for MnB strains were 77.3% and 81.1%, respectively, for A22; 94.4% and 95.3%, respectively, for A56; 63.0% and 66.0%, respectively, for B24; and 46.1% and 48.6%, respectively, for B44. The proportions of subjects achieving an hSBA titer fold rise ≥2 from baseline to 1 month after Vaccination 3 for Group 1 and Group 2 for MnB strains were 90.2% and 92.8%, respectively, for A22; 97.2% and 97.9%, respectively, for A56; 84.6% and 87.2%, respectively, for B24; and 77.7% and 81.7%, respectively, for B44.

The proportions of subjects achieving an hSBA titer fold rise ≥3 from baseline to 1 month after Vaccination 2 for Group 1 and Group 2 for MnB strains were 73.1% and 74.2%, respectively, for A22; 92.5% and 92.6%, respectively, for A56; 61.3% and 63.4%, respectively, for B24; and 45.7% and 47.4%, respectively, for B44. The proportions of subjects achieving an hSBA titer fold rise ≥3 from baseline to 1 month after Vaccination 3 for Group 1 and Group 2 for MnB strains were 85.3% and 86.4%, respectively, for A22; 95.0% and 95.3%, respectively, for A56; 83.4% and 84.8%, respectively, for B24; and 77.0% and 80.7%, respectively, for B44.

In summary of the descriptive endpoints under the objectives, the majority of subjects achieved an hSBA titer ≥LLOQ for both Group 1 (bivalent rLP2086+GARDASIL) and group 2 (bivalent rLP2086+saline) for all 4 primary MnB test strains, while only a very small proportion of subjects had measurable hSBA titers ≥LLOQ at baseline (prevaccination Visit 1). Substantial immune responses with the 4 MnB strains were observed at 1 month after Vaccination 2, with additional increases observed at 1 month after Vaccination 3 for both Group 1 and Group 2 subjects. This conclusion was confirmed by the proportion of subjects with an hSBA titer of ≥16 following 3 doses, the observed GMTs achieved after 2 doses and after 3 doses in both groups, and the RCDCs for the 4 primary MnB test strains.

For both Group 1 and Group 2, a high proportion of subjects achieved an hSBA titer fold rise ≥4 for each of the primary MnB test strains and a composite hSBA response ≥LLOQ for all 4 primary MnB strains after the third study vaccination.

In addition, the majority of subjects achieved an hSBA titer fold rise ≥3 and an hSBA titer fold rise ≥2 for the 4 primary MnB strains at all sampling time points for both Group 1 (bivalent rLP2086+GARDASIL) and Group 2 (bivalent rLP2086+saline). The proportion of subjects with results meeting these criteria was higher after 3 vaccinations compared with 2 vaccinations.

These results support the evidence that the immune response to bivalent rLP2086 when coadministered with the HPV vaccine, GARDASIL, yields a robust immune response that is comparable to the immune response to bivalent rLP2086+saline.

HPV GMTs. Table B of Example 8 presents the GMTs and the corresponding Cls for each of the 4 HPV antigens at 1 month after Vaccination 3 for Group 1 (bivalent rLP2086+GARDASIL) and Group 3 (saline+GARDASIL) in the evaluable immunogenicity population.

For Group 3, the HPV GMTs at baseline (before Vaccination 1) and at 1 month after Vaccination 3 were 6.0 and 550.3, respectively, for HPV-6; 4.3 and 1084.3, respectively, for HPV-11; 6.1 and 4763.4, respectively, for HPV-16; and 5.3 and 1047.4, respectively, for HPV-18. For Group 1 (bivalent rLP2086+GARDASIL), the HPV GMTs at baseline (before Vaccination 1) and at 1 month after Vaccination 3 were 5.8 and 451.8, respectively for HPV-6; 4.2 and 892.9, respectively, for HPV-11; 5.8 and 3695.4, respectively, for HPV-16; and 5.2 and 744.0, respectively, for HPV-18. Overall, the GMTs were numerically higher for Group 3 compared with Group 1. Reverse cumulative distribution curves (RCDCs) showing the distribution of titers for HPV-6, HPV-11, HPV-16, and HPV-18 were assessed for Group 1 (bivalent rLP2086+GARDASIL) and Group 3 (saline+GARDASIL) at all sampling time points for the evaluable immunogenicity population. The RCDCs showed robust immune responses among subjects after Vaccination 3 for both Group 1 and Group 3.

Summary of Immune response to GARDASIL. The GMTs to HPV antigens were numerically higher for Group 3 (saline+GARDASIL) as compared with Group 1 (bivalent rLP2086+GARDASIL), and the observed HPV GMTs after Vaccination 3 were indicative of a robust immune response for both groups. RCDCs also supported robust immune responses after Vaccination 3 for both Group 1 and Group 3. This was also supported by the proportion of subjects with seropositive status for the 4 HPV antigens, which was >99% at 1 month after Vaccination 3 for both groups. The younger age subgroup had higher HPV GMTs in Group 3 (saline+GARDASIL) than the older age subgroup. This difference was maintained when GARDASIL was given concomitantly with bivalent rLP2086.

Immunogenicity Conclusions. The noninferiority criteria of bivalent rLP2086_GARDASIL compared to saline+GARDASIL or compared to bivalent rLP2086+saline required that the lower limit of the 2-sided 95% Cls for the geometric mean titer ratios (GMRs) for antibodies to HPV for all 4 HPV antigens (HPV-6, HPV-11, HPV-16, and HPV-18) and for hSBA titers using 2 primary MnB test strains (A22 and B24) 1 month after Vaccination 3 be greater than 0.67. This prespecified threshold was met for both MnB strains and 3 of the 4 HPV antigens. For HPV-18, the lower limit of the 2-sided 95% Cls for the GMR was slightly below the prespecified threshold of 0.67, at 0.62.

Seroconversion for all 4 HPV antigens was achieved by 99% or more of the subjects for the groups that received GARDASIL concomitantly with bivalent rLP2086 or with saline. The RCDCs for all 4 HPV antigens show that the majority of subjects achieved a response above the seroconversion threshold at 1 month after Vaccination 3. Robust GMTs relative to baseline were observed for both groups that received GARDASIL.

The 4-fold rise responses to 2 primary MnB test strains (A22 and B24) were similar (ranged from 83.4% to 86.4%) between the group that received bivalent rLP2086+GARDASIL (85.3% and 83.4%, respectively) and the group that received bivalent rLP2086+saline (86.4% and 84.8%, respectively).

Further descriptive analyses of the response to bivalent rLP2086 were performed using 4 primary MnB test strains (A22, A56, B24, and B44). A high proportion of subjects achieved an hSBA titer fold rise ≥4 and the composite response (all 4 primary MnB test strains and the same immunogenicity/efficacy endpoint definition as used in the Phase 3 clinical program) for the evaluable immunogenicity population for both groups that received bivalent rLP2086, either concomitantly with GARDASIL (bivalent rLP2086+GARDASIL) or with saline (bivalent rLP2086+saline), 1 month after Vaccination 2 or 3. These responses are substantially higher than an hSBA titer ≥4 that has been demonstrated to correlate with protection against meningococcal disease including serogroup B disease. These results also indicate and support the evidence of a robust immune response to bivalent rLP2086 whether administered with saline or concomitantly with GARDASIL.

Conclusions: Data indicate that robust immune responses to both vaccines were generated after concomitant administration of rLP2086+HPV4. Prespecified noninferiority criteria were met for 5 of 6 antigens. Although GMRs to HPV-18 narrowly missed noninferiority criteria, the high proportion of responders (99%) indicates clinical effectiveness is expected to be maintained after concomitant administration. Bivalent rLP2086 was well tolerated and elicited a robust immune response to test strains expressing fHBPs heterologous to those in the vaccine.

TABLE A Comparison of Geometric Mean Titers at 1 Month After Vaccination 3 (Evaluable Immunogenicity Population) Group 1 Group 2 Group 3 Strain rL P2086 + HPV4 rL2086 + Saline Saline + HPV4 [Variant] n^(a) GMT^(b) (95% CI)^(c) n^(a) GMT^(b) (95% CI)^(c) n^(a) GMT^(b) (95% CI)^(c) Ratio^(d) (95% CI)^(e) HPV antigens (Group 1 vs Group 3) HPV-6 813 451.8 (417.5, 489.0) NA 423 550.3 (490.4, 617.6) 0.82 (0.72, 0.94) HPV-11 813 892.9 (839.5, 949.6) 423 1084.3 (997.3, 1179.0) 0.82 (0.74, 0.91) HPV-16 813  3695.4 (3426.3, 39857) 423  4763.4 (4285.9, 5294.2) 0.78 (0.68, 0.88) HPV-18 813 744.0 (687.7, 805.0) 423 1047.4 (939.0, 1168.3) 0.71 (0.62, 0.81) hSBA strains (Group 1 vs Group 2) PMB80 [A22] 803 53.3 (50.2, 56.7)  801 57.8 (54.4, 61.4) NA 0.92 (0.85, 1.00) PMB2948 [B24] 788 25.8 (24.1, 27.6)  793 28.0 (26.2, 29.9) 0.92 (0.84, 1.01) CI = confidence interval; GMT = geometric mean titer; HPV = human papillomavirus; hSBA = serum bactericidal assay using human complement; LLOQ = lower limit of quantitation; NA = not applicable. Note: LLOQ = 11 mMU/ml for HPV-6, 8 mMU/ml for HPV-11; 11 mMU/ml for HPV-16; and 10 mMU/ml for HPV-18. LLOQ = 1:16 for A22; 1:8 for A56, B24, and B44. Results below the LLOQ were set to 0.5* LLOQ for analysis. ^(a)n = number of subjects with valid and determinate assay results for the given antigen or strain. ^(b)Geometric mean titers (GMTs) were calculated using all subjects with valid and determinate assay results at 1 month after Vaccination 3. ^(c)Confidence intervals (CIs) are back transformations of confidence levels based on the Student t distribution for the mean logarithm of assay results. ^(d)Ratios of GMTs (Group 1/Group 3 for HPV antigen titers and Group 1/Group 2 for hSBA strain titers). ^(e)Confidence Intervals (CIs) for the ratio are back transformations of a confidence interval based on the Student t distribution for the mean difference of the logarithms of the measures (Group 1-Group 3 for HPV titers and Group 1-Group 2 for hSBA strain titers).

TABLE B Comparison of Subjects Achieving HPV Seroconversion at 1 Month After Vaccination 3 - Baseline HPV Seronegative Evaluable Immunogenicity Population Group 1 Group 3 Seropositive rLP2086 + HPV4 Saline + HPV4 Difference Antigen Criteria N^(a) n^(b) (%) (95% CI)^(c) N^(a) n^(b) (%) (95% CI)^(c) (%)^(d) (95% CI)^(e) HPV-6 ≥20 mMU/mL 802 797 (99.4) (98.6, 99.8) 414 411 (99.3) (97.9, 99.9) 0.1 (−0.9, 1.5) HPV-11 ≥16 mMU/mL 801 798 (99.6) (98.9, 99.9) 417 415 (99.5) (98.3, 99.9) 0.1 (−0.7, 1.3) HPV-16 ≥20 mMU/mL 800 797 (99.6) (98.9, 99.9) 413 411 (99.5) (98.3, 99.9) 0.1 (−0.7, 1.3) HPV-18 ≥24 mMU/mL 805 801 (99.5) (98.7, 99.9) 418 414 (99.0) (97.6, 99.7) 0.5 (−0.6, 1.9) CI = confidence interval; HPV = human papillomavirus. ^(a)N = number of subjects with baseline HPV seronegative status for the given antigen. ^(b)n = Number of subjects achieving seroconversion (prespecified criteria) at 1 month after Vaccination 3 for the given antigen. ^(c)Exact 2-sided confidence interval (Clopper and Pearson) based upon the observed proportion of subjects. ^(d)Difference in proportions, expressed as a percentage. ^(e)Exact 2-sided confidence interval (based on Chan & Zhang) for the difference in proportions, expressed as a percentage.

Example 9: Bivalent RLP2086 Vaccine Efficacy

The efficacy of bivalent rLP2086 has been inferred using hSBA responses as the surrogate of efficacy and demonstration of serum bactericidal antibody responses to invasive N. meningitidis serogroup B (MnB) strains.

Four MnB strains, representative of invasive meningococcal disease (IMD) causing strains, were used in the evaluation. Each MnB test strain expresses an fHBP protein variant (A22, A56, B24 or B44) that is heterologous (differs) from the vaccine components (A05 and B01).

The efficacy of bivalent rLP2086 was assessed in 3 randomized controlled Phase II studies conducted in 4,459 adolescents aged 11 through 18 years of age in the US and Europe. See also Example 6. A total of 2,293 received at least 1 dose of 120 μg of bivalent rLP2086 using a 0-, 2-, and 6-month vaccination schedule. Efficacy was assessed by evaluating hSBA immune responses in subjects vaccinated with bivalent rLP2086.

Efficacy was inferred using 5 co-primary immunogenicity endpoints. For 4 of the 5 co-primary endpoints, pre-specified proportions of subjects had to achieve 4-fold rises in hSBA titer to each of the 4 MnB test strains following 3 doses of bivalent rLP2086. The fifth co-primary endpoint was a composite endpoint requiring that a prespecified high proportion of subjects each respond in all 4 hSBAs with the primary MnB test strains following 3 doses of bivalent rLP2086. Immune response was also assessed based on the proportion of subjects who achieved an hSBA titer the lower limit of quantitation (LLOQ) 1 month after the third dose of vaccine. LLOQ is defined as the lowest amount of the antibody in a sample that can be measured.

Study 1 (described in Example 7 and Example 8) was a Phase II, randomized, active-controlled, observer-blinded, multicenter trial in which 2,499 US subjects, 11 through 17 years of age, were randomly assigned (in a 2:2:1 ratio) to 1 of 3 groups: Group 1 received bivalent rLP2086+HPV4, Group 2 received bivalent rLP2086+Saline, and Group 3 received Saline+HPV4. All vaccinations were administered on a 0-, 2-, and 6-month schedule.

Study 2 (described in Example 4) was a Phase II, randomized, placebo-controlled, single-blind trial in which 753 European subjects, 11 through 18 years of age, were randomly assigned in a 1:1 ratio to 2 groups: Group 1 received bivalent rLP2086 at 0-, 2-, and 6-months and dTaP-IPV (diphtheria, tetanus, acellular pertussis-inactivated polio virus) at Month 0. Group 2 received Saline at 0-, 2-, and 6-months and dTaP-IPV at Month 0.

Study 3 (described in Example 5) was a Phase II, randomized, placebo-controlled, single-blind, multicenter trial in which 1,713 European subjects, 11 through 18 years of age, were randomly assigned in a 3:3:3:2:1 ratio to 5 groups. Subjects received 2 or 3 doses of bivalent rLP2086 administered on a 0-, 1-, and 6-month schedule (Group 1); on a 0-, 2-, and 6-month schedule (Group 2); on a 0- and 6-month schedule (Group 3); on a 0- and 2-month schedule (Group 4); or on a 0- and 4-month schedule (Group 5). Saline injections (1 or 2 doses depending on group) were administered in each group to maintain the blind.

Results in Studies 1, 2, and 3 among subjects who received a 3-dose series of bivalent rLP2086 at 0-, 2-, and 6-months are described above in the respective Examples 4-8. Evaluation of the 4-fold and composite response rates were exploratory endpoints for all studies. The 4-fold response rates showed that the lower bounds of the 95% Confidence Interval (CI) for all 4 endpoints were similar among the 3 studies and consistently met the threshold limits for the Phase III endpoints. The proportion of subjects achieving hSBA titer ≥LLOQ was similar across the 3 studies.

Based on the hSBA data acquired following 2 administrations of the vaccine given 1 or 2 months apart, 2 doses of vaccine administered over these intervals may provide protection to individuals at increased risk, due to potential exposure to a case of meningococcal serogroup B disease. The responses observed after 2 vaccine administrations delivered 1 or 2 months apart showed that a proportion of subjects expressed hSBA levels equal to or above the LLOQ values for each of the 4 primary test strains (see Study 1 results for Group 1 and Group 2; see Study 2 results for Group 1; see Study 3 results for Group 2). A third dose of the vaccine, administered at 6 months, can achieve vaccine-mediated protection.

Concomitant Vaccine Administration. Study 1 (described in Example 7 and Example 8) evaluated the concomitant use of bivalent rLP2086 and HPV4 in US adolescents. The study endpoints included noninferiority assessment of the immune response for the four HPV4 antigens (based on geometric mean titer [GMT]) and for bivalent rLP2086 (based on hSBA using two MnB test strains [variants A22 and B24]) 1 month after the third vaccination. HPV4 immune response was also evaluated by seroconversion for each of the 4 HPV antigens.

Study 1 shows the comparison of the geometric mean titers (GMTs) of the antibodies to HPV antigens for Group 1 (bivalent rLP2086+HPV4) and Group 3 (Saline+HPV4), with their corresponding GMT ratio (GMRs) between Group 1 and Group 3 and the 2-sided 95% Cls of the ratios. Study 1 also provides the comparison of hSBA GMTs to the 2 primary MnB test strains for Group 1 and Group 2 with their corresponding GMRs between Group 1 and Group 2 and the 2-sided 95% CI of the ratios. The criterion for noninferiority margin was 1.5-fold, which corresponds to a value of 0.67 for the lower limit of the 2-sided 95% CI of the GMR. The 1.5-fold criterion of 0.67 was met for all the MnB test strains and the HPV antigens except for HPV-18, which had a lower bound 95% confidence interval (CI) of 0.62. Although the response to HPV-18 did not meet the pre-specified noninferiority criterion, the difference was marginal. In a separate analysis, ≥99%, of subjects seroconverted to all 4 HPV antigens in both the Saline+HPV4 and bivalent rLP2086+HPV4 groups.

Example 10: Bivalent rLP2086 Elicits Antibodies in Individuals that Provide Broad Coverage Against MnB Strains Expressing Prevalent and Outbreak-Associated fHBP Variants

Bactericidal antibodies measured in serum bactericidal assays using human complement (hSBAs) have been correlated with protection from meningococcal disease and hSBA responses have been used routinely as surrogates of vaccine efficacy. Global epidemiological studies of fHBP diversity revealed thatn ˜80% of meningococcal disease is caused by strains that express one of 10 prevalent fHBP variants.

Methods: hSBA responses to Neisseria meningitidis serogroup B (MnB) strains expressing the 10 most prevalent fHBP variants in the US and Europe (B24, B16, B44, A22, B03, B09, A12, A19, A05 and A07) in individual human subjects immunized with bivalent rLP2086 were evaluated. MnB strains expressing these ten most prevalent variants represent the breadth of fHBP diversity, including 5 of the 6 major fHBP subgroups, that are representative of >98% and 97% of strains (by subgroup) in the MnB SBA strain pool, and US subset of the MnB SBA strain pool, respectively. Twenty-three MnB test strains were obtained from Pfizer's MnB SBA strain pool (N=1263) that represent strains systematically collected from the US and Europe between the years 2000 and 2006. In addition, isolates from recent MnB disease outbreaks were included in the analysis. Matched prevaccination and postvaccination sera (postdose 2 and postdose 3) were obtained randomly from adolescent and young adult subjects enrolled in clinical studies B1971005, B1971012 or B1971003.

To provide additional information supporting the potential coverage afforded by vaccination with bivalent rLP2086, hSBAs were performed with the outbreak strains and serum samples from nine subjects immunized with bivalent rLP2086 (clinical study B1971012, described in Example 5 and Example 6. The subjects (11 to <19 years of age) had received 3 doses of bivalent rLP2086 at 0, 2 and 6 months. To ensure a conservative hSBA assessment the nine subjects were selected in a non-biased manner from a set of subjects with no baseline hSBA activity against the primary MnB test strains. Two of the clonal Princeton University outbreak strains (PMB5021 and PMB5025) and two of the UCSB outbreak strains (one from each of the two genetic clusters, PMB4478 and PMB4479, were tested.

Genetic characterization of the clonal Princeton University MnB Outbreak Strains is as follows: data suggest that the Princeton University outbreak strains are clonal. Each of the strains was typed as CC41/44 (ST 409) and expressed fHBP variant B153 (SEQ ID NO: 6). The strains had identical allele assignments for NHBA (2), porA (subtype P1.5-1, 2-2) and porB (3-82), all were null for nadA, and all had the same pulsed field gel electrophoresis (PFGE) profile (429).

Genetic characterization of the 2013 University of California Santa Barbara Outbreak Strains is as follows: The UCSB strains were typed as CC32(ET5; ST32), expressed fHBP variant B24, and are related to the Oregon clone that has been associated with hyperendemic serogroup B disease since 1993. Unlike the Princeton outbreak group of strains, the UCSB strains segregated genetically into two distinct clusters that were differentiated by their PFGE profile (468 or 467) and porB type (3-461 or 3-24). The strains had identical allele assignments for NadA (1), NHBA (5), porA (subtype P1.7, 16-20)

hSBA titers at baseline for all subjects and all outbreak strains were <4, indicating that the subjects had no protective antibodies to any of the outbreak strains prior to immunization with bivalent rLP2086.

Results: All 23 MnB strains were susceptible in hSBA with sera from individual subjects immunized with bivalent rLP2086. Strains representing all 10 prevalent fHBP variants as well as additional strains were all killed by hSBA. Baseline hSBA seroprotection rates (proportions of subjects achieving hSBA titers ≥4) were generally low. The lower seroprotective rates observed in subjects before immunization with bivalent rLP2086 exemplify the vulnerability of a non-vaccinated adolescent or young adult population to MnB disease. However, robust seroprotection rates were observed in adolescents and young adults with postvaccination sera: seroprotection rates >70% were observed for 83% of these strains depending on MnB strains and population tested. Postvaccination seroprotection rates for strains expressing the most prevalent subfamily A and B fHBP variants, B24 and A22, ranged from 81.0% to 100%, and 77.8% to 100% for recent outbreak strains expressing fHBP variants B24 and B153. Furthermore, robust postdose 2 responses (compared to baseline) to all outbreak strains were observed in these subjects, ranging from 56 to 89% depending on the outbreak strain used in the hSBA. In contrast, prevaccination seroprotective rates were low, or not detectable, for recent US outbreak strains. The hSBA responses to the Princeton University and UCSB outbreak strains are shown in FIG. 2.

Conclusions: Bivalent rLP2086 elicits robust seroprotective hSBA responses in individuals to diverse invasive MnB strains expressing prevalent fHBPs in the US and Europe, as well as newly emerging variants (B153)(SEQ ID NO: 6). The proportion of subjects that showed a seroprotective response after immunization with bivalent rLP2086 greatly exceeded the proportion of subjects that was seroprotected at baseline. The data support that bivalent rLP2086 has the potential to provide broad protection of adolescents and young adults from invasive meningococcal serogroup B disease, including disease from recent outbreaks.

>B153  (SEQ ID NO: 6) CSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAA QGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYK QSHSALTALQTEQVQDSEDSGKMVAKRQFRIGDIAGEHTSFDKLPKGGSA TYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAAAYIKP DEKHHAVISGSVLYNQDEKGSYSLGIFGGKAEEVAGSAEVKTVNGIRHIG LAAKQ

Example 11: Immunogenicity and Safety of Bivalent rLP2086, a Meningococcal Serogroup B Vaccine, Coadministered With Tdap and MCV4 in US Adolescents

A Phase 2, Randomized, Active-Controlled, Observer Blinded Trial, to Assess the Safety, Tolerability, and Immunogenicity of MCV4, Tdap Vaccine and Bivalent rLP2086 Vaccine When Administered Concomitantly in Healthy Subjects Aged ≥10 to <13 Years

Background: Concurrent administration of bivalent rLP2086 with vaccines recommended in the US may improve adherence to vaccine schedules.

Objective: To determine if immune responses induced by coadministration of MENACTRA® [meningococcal A, C, Y and W-135 polysaccharide conjugate vaccine (MCV4)] and ADACEL® [tetanus toxoid, reduced diphtheria toxoid, acellular pertussis vaccine (Tdap)] with TRUMENBA® [meningococcal B (MnB) vaccine], approved in the United States (bivalent rLP2086)] were noninferior to MCV4+Tdap or bivalent rLP2086 alone

Design/Methods: Healthy adolescents aged ≥10 to <13 y received

MCV4+Tdap+bivalent rLP2086, MCV4+Tdap, or bivalent rLP2086. Bivalent rLP2086 immunogenicity was assessed by serum bactericidal assay using human complement (hSBA) with 2 MnB test strains expressing vaccine-heterologous fHBP variants; multiplexed LUMINEX assays and/or rabbit SBAs assessed immunogenicity of MCV4/Tdap antigens. Safety was assessed.

Results: 2648 subjects received MCV4+Tdap+bivalent rLP2086, MCV4+Tdap, or bivalent rLP2086. Immune responses to MCV4, Tdap, and bivalent rLP2086 given concomitantly were noninferior to immune responses to MCV4+Tdap or bivalent rLP2086 alone. 62.3-68.0% and 87.5-90% of MCV4+Tdap+bivalent rLP2086 recipients after vaccination 2 and 3, respectively, had prespecified seroprotective hSBA titers to 2 MnB test strains; bivalent rLP2086 alone induced similar responses. Concomitant administration did not significantly increase local reactions or systemic events compared to bivalent rLP2086 alone.

Bivalent rLP2086 given concomitantly with MCV4+Tdap met all noninferiority immunogenicity criteria without a clinically significant increase in reactogenicity. Convenience associated with concomitant administration may improve adherence to recommended schedules.

This study (B1971015) assessed the immunogenicity of quadrivalent meningococcal polysaccharide conjugate (MCV4) vaccine; tetanus, diphtheria, and acellular pertussis (Tdap) vaccine; and bivalent rLP2086 when administered concomitantly. It also assessed the safety, tolerability and immunogenicity of bivalent rLP2086 in healthy subjects aged ≥10 to <13 years in the United States. The 2 MnB test strains used in hSBA to evaluate noninferiority of the immune response to bivalent rLP2086 when given with or without the licensed vaccines express the two most prevalent fHBPs in the United States, namely A22 and B24.

Saline was used as a placebo to maintain the study blind because there was no proven safe, immunogenic, and effective vaccine against MnB that could serve as an active control. In addition, saline was chosen as an inert control as opposed to a control vaccine with excipients (such as aluminum) because the safety objective to describe the tolerability profile of the investigational product was met with saline rather than another vaccine.

Approximately 2625 subjects were to participate in this study at approximately 88 sites in the United States (approximately 30 subjects at each site). Subjects were randomly assigned to 1 of 3 groups in a 1:1:1 ratio (Group 1:Group 2:Group 3) (Table A). Subjects received investigational products at each of the vaccination visits (Visits 1, 3, and 5).

Subjects presenting with any of the following were not to be included in the study: Previous vaccination with any meningococcal serogroup B vaccine; Vaccination with any diphtheria, tetanus or pertussis vaccine within 5 years of the first study vaccination; Previous vaccination with any MCV4 vaccine.

TABLE A Study Design Approx Month 0 1 2 3 6 7 12 Visit 1 Visit 2 Visit 3 Visit 4 Visit 5 Visit 6 Visit 7 Vaccination MCV4 + rLP2086 rLP2086 Final (Group 1) Tdap + telephone rLP2086 contact Vaccination MCV4 + Saline Saline Final (Group 2) Tdap + telephone Saline contact Vaccination Saline + rLP2086 rLP2086 MCV4 + Final (Group 3)^(a) Saline + Tdap^(b) telephone rLP2086 contact Blood draw ~20 mL ~20 mL ~20 mL ~20 mL (all groups) Abbreviation: rLP2086 = bivalent recombinant lipoprotein 2086 vaccine. ^(a)Subjects in Group 3 who withdrew or discontinued early should have been contacted to return to clinic to receive MCV4 and Tdap vaccinations. ^(b)Study sites were able to determine whether a subject was in Group 3 or not at Visit 6 (1 month after the last dose of either saline or rLP2086) in order to identify individuals who required MCV4 + Tdap. Subject assignment to Group 1 and 2 remained blinded throughout the study.

During the study, subjects in Groups 1 and 3 received bivalent rLP2086 at approximately 0, 2, and 6 months (Visits 1, 3, and 5, respectively) in the left arm. For subjects in Group 2, saline was administered by intramuscular (IM) injection (0.5 mL) at approximately 0, 2, and 6 months (Visits 1, 3, and 5, respectively) in the left arm. Subjects in Group 3 received 2 IM saline injections at 0 months in the right arm. MCV4 and Tdap vaccines were administered once only by IM injection at 0 months (Groups 1 and 2) in the right arm. Following the last blood draw at Visit 6 (post-Vaccination 3 blood draw), subjects in Group 3 received the MCV4 and Tdap vaccines.

MCV4 and Tdap vaccines were administered once only by IM injection at 0 months (Groups 1 and 2) into the upper deltoid muscle of the right arm. The vaccines were administered at least 5 cm apart. Subjects in Group 3 received MCV4 and Tdap vaccines at Visit 6 following their last blood draw.

Blood samples (approximately 20 ml each) were collected immediately before Vaccination 1, 28 to 41 days after Vaccination 1, 28 to 42 days after Vaccination 2, and 28 to 42 days after Vaccination 3 for immunogenicity analyses.

The bivalent rLP2086 vaccine used was in a 0.5 mL dose formulated to contain 60 μg each of a purified subfamily A rLP2086 protein (variant A05, SEQ ID NO: 1) and a purified subfamily B rLP2086 protein (variant B01, SEQ ID NO: 2), 0.15 M sodium chloride, 2.8 molar ratio polysorbate 80, and 0.25 mg of Al³+ as aluminum phosphate (AIPO4) as a stabilizer in 10 mM histidine-buffered saline at pH 6.0.

The MCV4 vaccine was MENACTRA® [meningococcal (Groups A, C, Y and W-135) polysaccharide diphtheria toxoid conjugate vaccine (MCV4)], a commercially available vaccine. MENACTRA is a solution supplied as a 0.5 mL dose for IM injection.

The vaccine contains N. meningitidis serogroup A, C, Y and W-135 capsular polysaccharide antigens individually conjugated to diphtheria toxoid protein. The four meningococcal components, present as individual serogroup-specific glycoconjugates, compose the final formulated vaccine. No preservative or adjuvant is added during manufacture. Each 0.5 mL dose could contain residual amounts of formaldehyde of less than 2.66 μg (0.000532%), by calculation. MENACTRA is manufactured as a sterile, clear to slightly turbid liquid. Each 0.5 mL dose of vaccine is formulated in sodium phosphate buffered isotonic sodium chloride solution to contain 4 μg each of meningococcal A, C, Y and W-135 polysaccharides conjugated to approximately 48 μg of diphtheria toxoid protein carrier.

The Tdap vaccine was ADACEL® (tetanus toxoid, reduced diphtheria toxoid and acellular pertussis vaccine adsorbed [Tdap]), a commercially available vaccine. ADACEL is supplied as a 0.5 mL dose for IM injection. The vaccine is a sterile isotonic suspension of tetanus and diphtheria toxoids and pertussis antigens adsorbed on aluminum phosphate. Each 0.5 mL dose contains 5 Lf tetanus toxoid, 2 Lf diphtheria toxoid, and acellular pertussis antigens (2.5 μg detoxified pertussis toxin, 5 μg filamentous hemagglutinin, 3 μg pertactin [PRN], 5 μg fimbriae types 2 and 3). Other ingredients per 0.5 mL dose include 1.5 mg aluminum phosphate (0.33 mg aluminum) as the adjuvant, ≤5 μg residual formaldehyde, <50 ng residual glutaraldehyde and 3.3 mg (0.6% v/v) 2-phenoxyethanol (not as a preservative). ADACEL does not contain a preservative.

Sterile normal saline solution for injection (0.9% sodium chloride) was in a 0.5 mL dose.

Bivalent rLP2086 Serum Bactericidal Assays. For assessment of the immune response to bivalent rLP2086, functional antibodies were analyzed in validated hSBAs. The hSBA measures antibodies in human sera that result in complement mediated killing of the target MnB strain. Sera obtained from all subjects in Groups 1 and 3, prior to the first vaccination with bivalent rLP2086, approximately 1 month after the second vaccination with bivalent rLP2086 (Visit 3), and approximately 1 month after the third vaccination with bivalent rLP2086 (Visit 5), were tested in hSBAs with strains PMB80 [A22] and PMB2948 [B24].

MCV4 Concomitant Vaccine SBAs. For assessment of the immune response to the MCV4 vaccine, functional antibodies were analyzed in serum bactericidal assays using rabbit complement (rSBAs) with meningococcal strains representing serogroups A, C, Y and W-135. Sera obtained from all subjects in Groups 1 and 2 prior to the first vaccination with MCV4 (Visit 1) and 1 month after vaccination with MCV4 (Visit 2) were used in these assays.

Antigen Detection Assays for MCV4 and Other Concomitant Vaccines.

Assessments of diphtheria, tetanus, and pertussis antibody responses were performed using a validated and multiplexed LUMINEX multiplex assay (MCV4 LXA). Sera obtained from all subjects in Groups 1 and 2 prior to the first vaccination with MCV4 and Tdap (Visit 1) and 1 month after vaccination with MCV4 and Tdap (Visit 2) were used in the assay. Additional assessments of meningococcal serogroups A, C, Y and W-135 responses were performed using a validated multiplexed LXA (A,C,Y,W LXA). Sera obtained from all subjects in Groups 1 and 2 prior to the first vaccination with MCV4 and Tdap (Visit 1) and 1 month after vaccination with MCV4 and Tdap (Visit 2) were used in these assays.

Sample size. The antibody responses to the 10 antigens in the marketed vaccines and 2 MnB strains were assumed to be independent. The criteria for the noninferiority margin were 1.5-fold.

Methods of Analysis. The LLOQ for PMB80 (A22) was 1:16 and the LLOQ for PMB2948 (B24) was 1:8. The LLOQs for Tdap/MCV4 assays are listed in Table B.

TABLE B LLOQs for Assays to determine immune responses to Marketed Vaccine Antigens Vaccine-Assay/Antigen LLOQ MCV4-rSBA Meningococcal serogroup A 1:32 Meningococcal serogroup C 1:64 Meningococcal serogroup Y  1:128 Meningococcal serogroup W-135 1:64 MCV4-LXA (IgG) Meningococcal serogroup A 0.1795 ug/mL^(a) Meningococcal serogroup C 0.0940 ug/mL^(b) Meningococcal serogroup Y 0.2485 ug/mL^(c) Meningococcal serogroup W-135 0.2530 ug/mL^(d) Tdap-LXA Diphtheria 0.037 IU/ml Tetanus 0.05 IU/ml Pertussis toxoid 0.9 IU/ml Pertussis filamentous haemagglutinin 2.9 EU/ml Pertussis pertactin 3.0 EU/ml Pertussis fimbriae types 2 + 3 10.6 EU/ml Abbreviations: IgG = immunoglobulin G; LLOQ = lower limit of quantitation; LXA = LUMINEX assay; MCV4 = quadrivalent meningococcal polysaccharide conjugate; rSBA = serum bactericidal assay using rabbit complement; Tdap = tetanus, diphtheria, and acellular pertussis. ^(a)Postvalidation LLOQ presented in table; validation LLOQ was 0.1037 μg/mL. ^(b)Postvalidation LLOQ presented in table; validation LLOQ was 0.0204 μg/mL. ^(c)Postvalidation LLOQ presented in table; validation LLOQ was 0.1104 [g/mL. ^(d)Postvalidation LLOQ presented in table; validation LLOQ was 0.1198 μg/mL. Analysis for Coprimary Objectives (Noninferiority Objectives)

GMTs and GMCs. For MnB strains PMB80 (A22) and PMB2948 (B24), hSBA titers measured at each blood sampling time points (Visits 1, 4, and 6) were logarithmically transformed for analysis and GMTs were computed for each primary strain and for each group at each blood sampling time point along with 2-sided 95% confidence intervals (Cis). The Cls were constructed by back transformation of the confidence limits computed for the mean of the logarithmically transformed assay data based on Student's t distribution. The GMTs determined in rSBAs to each of the 4 MCV4 antigens and the GMCs determined in LXAsr to each of the 4 MCV4 antigens were summarized similarly at Visit 1 and Visit 2. The GMC determined by LXA to each of the 6 antigens in the Tdap vaccine were summarized at Visit 1 and Visit 2.

Geometric Mean Ratios. For the purpose of testing the noninferiority hypotheses, geometric mean ratios (GMRs) between corresponding groups at each applicable visit (Group 1/Group 3 for the hSBA titer at Visit 6 and Group 1/Group 2 for the MCV4/Tdap antigens at Visit 2), along with 95% CI were used. The mean difference of the logarithmically transformed results was equivalent to the mean of the ratio on the logarithmic scale: log (Group 1/Group 2)=log(Group 1)−log (Group 2). The proper exponential transformation was used to calculate the GMR. Similarly, the 2-sided, 95% Cls for the ratios were constructed by back transformation of the Cls for the mean difference of the logarithmically transformed assay results computed using Student's t distribution. Statistical inference was based on the Cls of the GMRs (ie, lower bound of the 95% CI was >0.67).

Noninferiority for Marketed Vaccine (Coprimary for MCV4/Tdap). For the first coprimary objective, antibody titers or concentrations to each of the 10 antigens inthe marketed vaccines measured at 1 month after vaccination (Visit 2) were logarithmically transformed for analysis and GMs were computed for Group 1 and Group 2. The 2-sided 95% CI of the ratio ([bivalent rLP2086+MCV4/Tdap]/[MCV4/Tdap]) of GMs, were provided for each of the 10 antigens.

Noninferiority for rLP2086 Vaccine (Coprimary for bivalent rLP2086). For the second coprimary objective, the hSBA titers for each of the 2 primary strains measured at Visit 6 were logarithmically transformed for analysis and GMTs were computed for Group 1 and Group 3. The 2-sided 95% CI of the GMRs ([bivalent rLP2086+MCV4/Tdap]/bivalent rLP2086) were provided for each of the 2 primary strains. The study coprimary objectives on noninferiority of both marketed vaccines (MCV4/Tdap) and investigational product (bivalent rLP2086) was achieved when the lower limit of the 2-sided 95% CI for the GMRs at Visit 2 for ([bivalent rLP2086+MCV4/Tdap]/[MCV4/Tdap], and at Visit 6 for [bivalent rLP2086+MCV4/Tdap]/bivalent rLP2086), were greater than 0.67 for all 10 MCV4/Tdap antigens and each of the 2 primary strains. GMRs (Group 1/Group2) were descriptively summarized with 2-sided 95% CI, for IgG concentrations to each of the 4 antigens in MCV4 at Visit 2.

Analysis of hSBA Data. The number and proportions of subjects who achieved hSBA titers ≥LLOQ, ≥4, ≥8, ≥16, ≥32, ≥1:64, and ≥128 at each blood sampling time point, and with at least 2-fold, 3-fold, and 4-fold rise in hSBA titer from baseline to after Vaccination 2 and after Vaccination 3, were descriptively summarized along with the exact 2-sided 95% CI (or Clopper-Pearson confidence limit) of the proportion for the primary MnB test strains PMB80 (A22) and PMB2948 (B24) for Group 1 and Group 3. The exact CI for the proportion was computed using the F distribution. As an additional analysis for noninferiority of bivalent rLP2086, the hSBA 4-fold response rate for the A22 variant (PMB80) and B24 variant (PMB2948) were compared between Group 1 and Group 3. The difference of the rates (bivalent rLP2086/MCV4/Tdap-bivalent rLP2086 or Group 1-Group 3) and exact 2-sided 95% Cls on the difference were provided. The exact Cls were computed using the standardized test statistic and gamma=0.000001. This was considered a descriptive analysis.

Analysis of MCV4/Tdap Immunogenicity. The proportion of subjects who achieved a seroresponse to each MCV4/Tdap antigen, and the proportion of subjects who achieved an antibody level 1.0 IU/mL for tetanus and diphtheria toxoids were summarized with exact 2-sided 95% Cls for Group 1 and Group 2. Noninferiority with respect to seroresponse rates to each MCV4/Tdap antigen and the proportion of subjects achieving an antibody level 1.0 IU/mL for tetanus and diphtheria toxoid (Group 1-Group 2) were analyzed.

Immune Response to Bivalent rLP2086 and MCV4/Tdap Antigens When Administered Concomitantly. Noninferiority Analyses of Geometric Mean Concentrations and Geometric Mean Titers to Tdap and MCV4 Antigens. The first coprimary objective of this study was to demonstrate that the immune response based on GMTs (or GMCs where applicable) induced by MCV4 and Tdap vaccines given with bivalent rLP2086 (Group 1) was noninferior to the immune response induced by MCV4 and Tdap vaccines alone (Group 2) when measured 1 month after the first vaccination (Visit 2) in both groups. The primary endpoints for the first coprimary objective were the GMTs or GMCs to each of the 10 antigens in the marketed vaccines at 1 month after the first vaccination (Visit 2), among subjects in Groups 1 and 2.

The comparison of GMTs (or GMCs where applicable) to the 10 MCV4 and Tdap antigens for Group 1 and Group 2, with their corresponding GMT (or GMC where applicable) ratios (GMRs) of Group 1 to Group 2 and the 2-sided 95% CI of the ratios, is presented in Table C. The criterion for the noninferiority margin was 1.5-fold, which corresponds to a value of 0.67 for the lower limit of the 2-sided 95% CI of the GMR.

For the evaluable immunogenicity population, GMRs of Group 1 to Group 2 at 1 month after Vaccination 1 for the Tdap antigens were 0.94 for diphtheria toxoid (95% Cl: 0.86, 1.03), 0.92 for tetanus toxoid (95% Cl: 0.85, 0.99), 0.93 for pertussis toxoid (95% Cl: 0.85, 1.02), 0.91 for pertussis filamentous hemagglutinin (95% Cl: 0.84, 0.98), 0.88 for pertussis pertactin (95% Cl: 0.80, 0.98), 0.90 for pertussis fimbrial agglutinogens types 2+3 (95% Cl: 0.74, 1.08); and the GMRs for the MCV4 antigens were 0.91 for serogroup A (95% Cl: 0.82, 1.01), 1.02 for serogroup C (95% Cl: 0.90, 1.15), 0.99 for serogroup Y (95% Cl: 0.89, 1.09), and 0.93 for serogroup W-135 (95% Cl: 0.83, 1.04). Therefore, the lower limits of the 2-sided 95% Cls for MCV4 and Tdap antigen GMRs for Group 1 compared to Group 2 were 0.86 for diphtheria toxoid, 0.85 for tetanus toxoid, 0.85 for pertussis toxoid, 0.84 for pertussis filamentous hemagglutinin, 0.80 for pertussis pertactin, 0.74 for pertussis fimbrial agglutinogens types 2+3, 0.82 for serogroup A, 0.90 for serogroup C, 0.89 for serogroup Y, and 0.83 for serogroup W-135. The 1.5-fold criterion of 0.67 (the lower limit of the 2-sided 95% CI of the GMR) was met for all MCV4 and Tdap antigens. Therefore, the first coprimary objective was met.

The immune response to the Tdap+MCV4 antigens when these vaccines are given concomitantly with bivalent rLP2086 was noninferior to the immune response obtained when they were given alone.

TABLE C Primary Immunogenicity Analysis - Comparison of Geometric Means at 1 Month After Last Vaccination - Evaluable Immunogenicity Populations Vaccine Group (as Randomized) Group 1 Group 2 Group 3 MCV4 + Tdap + rLP2086 MCV4 + Tdap + Saline Saline + Saline + rLP2086 Antigen/Strain (Variant) N^(a) GM^(b) (95% CI)^(c) N^(a) GM^(b) (95% CI)^(c) N^(a) GM^(b) (95% CI)^(c) Ratio^(d) (95% CI)^(e) Tdap antigens Diphtheria 778 9.3 (8.67, 9.92) 780 9.8 (9.23, 10.51) N/A N/A N/A 0.94 (0.86, 1.03) Tetanus 778 9.4 (8.95, 9.98) 780 10.3 (9.75, 10.85) N/A N/A N/A 0.92 (0.85, 0.99) Pertussis toxoid 778 13.2 (12.35, 14.14) 780 14.2 (13.28, 15.20) N/A N/A N/A 0.93 (0.85, 1.02) Pertussis filamentous 778 112.0 (106.15, 118.14) 780 122.9 (116.42, 129.84) N/A N/A N/A 0.91 (0.84, hemagglutinin 0.98) Pertussis pertactin 778 202.0 (187.77, 217.25) 780 228.9 (212.72, 246.35) N/A N/A N/A 0.88 (0.80, 0.98) Pertussis fimbriae 778 138.1 (121.20, 157.33) 780 154.2 (135.30, 175.79) N/A N/A N/A 0.90 (0.74, agglutinogens types 2 + 3 1.08) rSBA MCV4 antigens Serogroup A 763 4647.3 (4317.66, 772 5113.0 (4748.73, N/A N/A N/A 0.91 (0.82, 5002.09) 5505.17) 1.01) Serogroup C 768 1679.2 (1539.63, 767 1650.2 (1519.01, N/A N/A N/A 1.02 (0.90, 1831.38) 1792.65) 1.15) Serogroup Y 771 2212.6 (2056.08, 770 2244.9 (2088.70, N/A N/A N/A 0.99 (0.89, 2381.08) 2412.89) 1.09) Serogroup W-135 751 5925.1 (5469.77, 765 6367.9 (5872.68, N/A N/A N/A 0.93 (0.83, 6418.33) 6904.88) 1.04) hSBA MnB strains PMB80 (A22) 679 45.9 (42.74, 49.35) N/A N/A N/A 674 49.7 (46.43, 0.92 (0.84, 53.30) 1.02) PMB2948 (B24) 670 24.8 (23.11, 26.58) N/A N/A N/A 656 27.4 (25.58, 0.90 (0.82, 29.41) 1.00) Abbreviations: ELISA = enzyme-linked immunosorbent assay; EU = ELISA units; GM = geometric mean; hSBA = serum bactericidal assay using human complement; LLOQ = lower limit of quantitation; MCV4 = quadrivalent meningococcal polysaccharide conjugate (vaccine); MnB = Neisseria meningitidis serogroup B; N/A = not applicable; rSBA = serum bactericidal assay using rabbit complement; Tdap = tetanus, diphtheriam, and acellular pertussis (vaccine). Note: In order for the primary study hypothesis to be a success, the lower bound of the 95% CI of GM ratios for Tdap/MCV4 antigens and MnB strains must be >0.67. Note: For Tdap and MCV4 antigens, the post-Vaccination 1 evaluable immunogenicity population was used; for the MnB strains, the post-Vaccination 3 immunogenicity population was used. The Tdap/MCV4 antigens were not tested for Group 3 subjects and the MnB strains were not tested for Group 2 subjects. Note: LLOQ = 0.037 IU/mL for diphtheria; 0.05 IU/mL for tetanus; 0.9 EU/mL for pertussis toxoid; 2.9 EU/mL for pertussis filamentous hemagglutinin; 3.0 EU/mL for pertussis pertactin; 10.6 EU/mL for pertussis fimbrial agglutinogens types 2 + 3. LLOQ = 1:32 for MCV4 serogroup A, 1:64 for serogroup C, 1:128 for serogroup Y, and 1:64 for serogroup W-135. LLOQ = 1:16 for A22; 1:8 for B24. Results below the LLOQ were set to 0.5 × LLOQ for analysis. ^(a)N = number of subjects with valid and determinate assay results for the given antigen or strain. ^(b)GMs were calculated using all subjects with valid and determinate assay results at 1 month after last vaccination (Visit 6 for hSBA results and Visit 2 for all other results). ^(c)Confidence Intervals (CIs) are back transformations of confidence levels based on Student t distribution for the mean logarithm of assay results. ^(d)Ratios of GMs (Group 1/Group 2 for Tdap/MCV4 antigens and Group 1/Group 3 for MnB strains). ^(e)CIs for the ratio are back transformations of a confidence interval based on the Student t distribution for the mean difference of the logarithms of the measures (Group 1-Group 2 for Tdap/MCV4 antigens and Group 1-Group 3 for MnB strains). Noninferiority Analysis for hSBA GMTs to Bivalent rLP2086.

The second coprimary objective of this study was to demonstrate that the hSBA response to primary strains PMB80 (A22) and PMB2948 (B24) (based on GMTs) induced by MCV4+Tdap+bivalent rLP2086 (Group 1) was noninferior to the immune response induced by the saline+saline+bivalent rLP2086 (Group 3). The hSBA was performed 1 month after the third vaccination (Visit 6) with bivalent rLP2086 for both groups. The primary endpoints for the second coprimary objective were the evaluation of hSBA GMTs to each of the 2 primary strains (PMB80 [A22] and PMB2948 [B24]) measured at 1 month after the third dose of bivalent rLP2086 (Visit 6), which was administered to subjects in Group 1 (MCV4+Tdap+bivalent rLP2086) and Group 3 (saline+saline+bivalent rLP2086).

Table C presents the comparison of hSBA GMTs to the 2 primary MnB test strains for Group 1 and Group 3 with their corresponding GMRs of Group 1 to Group 3 and the 2-sided 95% Cls of the ratios, for the evaluable immunogenicity population. The criterion for the noninferiority margin was 1.5-fold, which corresponds to a value of 0.67 for the lower limit of the 2-sided 95% CI of the GMR.

For the evaluable immunogenicity population, the hSBA GMTs to the 2 primary MnB strains at 1 month after Vaccination 3 (bivalent rLP2086) for Group 1 and Group 3 were as follows: 45.9 and 49.7 respectively for PMB80 (A22) and 24.8 and 27.4, respectively for PMB2948 (B24). The GMRs of the MCV4+Tdap+bivalent rLP2086 group to the saline+saline+bivalent rLP2086 group at 1 month after Vaccination 3 (bivalent rLP2086) were 0.92 for PMB80 (A22) (95% Cl: 0.84, 1.02) and 0.90 for PMB2948 (B24) (95% Cl: 0.82, 1.00). The lower limits of the 2-sided 95% Cls for the hSBA GMRs for Group 1 compared to Group 3 were 0.84 for PMB80 (A22) and 0.82 for PMB2948 (B24), which are both greater than 0.67 and the noninferiority margin of 1.5-fold was met. Therefore, the second coprimary objective was achieved.

The immune response to the bivalent rLP2086 vaccine when given concomitantly with Tdap+MCV4 was noninferior to the immune response obtained when the vaccine was given alone.

Noninferiority Analysis for the Coprimary Objectives.

The first coprimary objective for the noninferiority of bivalent rLP2086 given with MCV4 and Tdap vaccines compared to both MCV4 and Tdap vaccines alone was tested by examining a noninferiority margin set at 1.5 fold, which corresponds to a value of 0.67 for the lower limit of the 2-sided 95% CI of the GMR for each of the 10 MCV4 and Tdap antigens for the post-Vaccination 1 evaluable immunogenicity population. This criterion was met for all MCV4 and Tdap antigens tested.

The second coprimary objective for the noninferiority of bivalent rLP2086 given with MCV4 and Tdap vaccines compared to the bivalent rLP2086 alone was tested by examining a noninferiority margin set at 1.5 fold, which corresponds to a value of 0.67 for the lower limit of the 2-sided 95% CI of the GMR for each of the 2 primary MnB strains (variants A22 and B24) in the post-Vaccination 3 evaluable immunogenicity population when measured 1 month after Vaccination 3. This criterion was met for both MnB test strains PMB80 (A22) and PMB2948 (B24).

Therefore, both coprimary objectives were achieved at the 1.5 fold noninferiority margin for all antigens in Tdap, MCV4, and bivalent rLP2086 vaccines.

Description of MCV4/Tdap Seroresponse Rates. One of the secondary objectives of this study was to describe seroresponses to antigens contained in MCV4 and Tdap (Groups 1 and 2) when measured 1 month after vaccination with MCV4 and Tdap (Visit 2).

The seroresponse rates for the 10 Tdap and MVC4 antigens with the corresponding 95% Cls in each group, and the percent differences (Group 1-Group 2), and the 95% Cls of the differences are presented in Table D for the post-Vaccination 1 evaluable immunogenicity population.

For the evaluable immunogenicity population, the seroresponse rates for the 6 Tdap antigens at 1 month after Vaccination 1 for Group 1 and Group 2 were as follows: 98.6% and 98.3%, respectively (diphtheria toxoid); 97.7% and 97.4%, respectively (tetanus toxoid); 68.1 and 72.7%, respectively (pertussis toxoid); 85.3% and 89.2%, respectively (pertussis filamentous hemagglutinin); 96.0% and 96.2%, respectively (pertussis pertactin); and 79.5% and 81.9%, respectively (pertussis fimbrial agglutinogens types 2+3). For the evaluable immunogenicity population, the seroresponse rates for the 4 MCV4 antigens at 1 month after Vaccination 1 for Group 1 and Group 2 were as follows: 85.4% and 88.6%, respectively (serogroup A); 89.3% and 88.9%, respectively (serogroup C); 90.5% and 93.6%, respectively (serogroup Y); and 97.1% and 97.2%, respectively (serogroup W-135). The difference in seroresponse rates of Group 1 to Group 2 at 1 month after Vaccination 1 were 0.2% for diphtheria toxoid (95% Cl: (−1.1, 1.6), 0.2% for tetanus toxoid (95% Cl: −1.4, 1.8), −4.6% for pertussis toxoid (95% Cl: −9.1, −0.1), −4.0% for pertussis filamentous hemagglutinin (95% Cl: −7.3, −0.6), −0.2% for pertussis pertactin (95% Cl: −2.1, 1.8), −2.5% for pertussis fimbrial agglutinogens types 2+3 (95% Cl: −6.4, 1.5), −3.2% for serogroup A (95% Cl: −6.7, 0.3), 0.3% for serogroup C (95% Cl: −2.8, 3.5), −3.2% for serogroup Y (95% Cl: −6.1, −0.4), and −0.1% for serogroup W-135 (95% Cl: −1.9, 1.7).

For the MCV4+Tdap+bivalent rLP2086 group (Group 1) and the MCV4+Tdap+saline group (Group 2), the estimated seroresponse rate differences were between −4.6% and 0.3% for the 6 Tdap and 4 MCV4 antigens, with the lower bounds of the 95% CI of the rate difference −9.1% or greater (ie, all greater than −10%).

TABLE D Comparisons of Seroresponse Rates for Tdap/MCV4 Antigens at 1 Month After Vaccination 1 - Post- Vaccination 1 Evaluable Immunogenicity Population Vaccine Group (as Randomized) Group 1 Group 2 Tdap/MCV4 MCV4 + Tdap + rLP2086 MCV4 + Tdap + Saline Difference Antigen N^(a) n^(b) (%) (95% CI)^(c) N^(a) n^(b) (%) (95% CI)^(c) (%)^(d) (95% CI)^(e) Tdap Diphtheria 774 763 (98.6) (97.5, 99.3) 780 767 (98.3) (97.2, 99.1) 0.2 (−1.1, 1.6) Tetanus 774 756 (97.7) (96.3, 98.6) 780 760 (97.4) (96.1, 98.4) 0.2 (−1.4, 1.8) Pertussis toxoid 774 527 (68.1) (64.7, 71.4) 780 567 (72.7) (69.4, 75.8) −4.6  (−9.1, −0.1) Pertussis filamentous hemagglutinin 774 660 (85.3) (82.6, 87.7) 780 696 (89.2) (86.8, 91.3) −4.0  (−7.3, −0.6) Pertussis pertactin 774 743 (96.0) (94.4, 97.3) 780 750 (96.2) (94.6, 97.4) −0.2 (−2.1, 1.8) Pertussis fimbriae agglutinogens types 2 + 3 774 615 (79.5) (76.4, 82.3) 780 639 (81.9) (79.0, 84.6) −2.5 (−6.4, 1.5) rSBA MCV4 Serogroup A 712 608 (85.4) (82.6, 87.9) 736 652 (88.6) (86.1, 90.8) −3.2 (−6.7, 0.3) Serogroup C 738 659 (89.3) (86.8, 91.4) 742 660 (88.9) (86.5, 91.1) 0.3 (−2.8, 3.5) Serogroup Y 754 682 (90.5) (88.1, 92.5) 753 705 (93.6) (91.6, 95.3) −3.2  (−6.0, −0.4) Serogroup W-135 729 708 (97.1) (95.6, 98.2) 752 731 (97.2) (95.8, 98.3) −0.1 (−1.9, 1.7) Abbreviations: ELISA = enzyme-linked immunosorbent assay; EU = ELISA units; LLOQ = lower limit of quantitation; MCV4 = quadrivalent meningococcal polysaccharide conjugate (vaccine); rSBA = serum bactericidal assay using rabbit complement; Tdap = tetanus, diphtheria, and acellular pertussis (vaccine). Note: Seroresponse is defined as: (1) For Tdap antigens, ≥4-fold rise in antibody concentration if the prevaccination antibody concentration is ≤ the cutoff value, and ≥2-fold rise in antibody concentration if the prevaccination antibody concentration is > the cutoff value. (2) For MCV4, seroresponse is defined as ≥4-fold rise on rSBA titers if baseline value is ≥ LLOQ, and postdose rSBA titers ≥2 × LLOQ if baseline value is < LLOQ. Note: Cutoff value = 0.1 IU/mL for diphtheria; 0.1 IU/mL for tetanus; 0.9 EU/mL for pertussis toxoid; 2.9 EU/mL for pertussis filamentous hemagglutinin; 3.0 EU/mL for pertussis pertactin; 10.6 EU/mL for pertussis fimbriae agglutinogens types 2 + 3. LLOQ = 1:32 for MCV4 serogroup A, 1:64 for serogroup C, 1:128 for serogroup Y, and 1:64 for serogroup W-135. ^(a)N = number of subjects with valid and determinate assay results for the given antigen at both the specified time point and baseline. ^(b)n = Number of subjects with seroresponse. ^(c)Exact 2-sided confidence intervals (CIs) based upon observed proportion of subjects using the Clopper and Pearson method. ^(d)Difference in proportions, expressed as a percentage. ^(e)Exact 2-sided CI (based on Chan and Zhang) for the difference in proportions, expressed as a percentage.

Description of hSBA Titer 4-Fold Rise. MCV4+Tdap+bivalent rLP2086 (Group 1) was compared to saline+saline+bivalent rLP2086 (Group 3), by analyzing the hSBA titer 4-fold response rates for 2 primary MnB strains (PMB80 [A22] and PMB2948 [B24]) at 1 month after Vaccination 3. The number and proportion of subjects achieving an hSBA titer fold rise ≥4 from baseline to 1 month after Vaccination 3 for the 2 primary MnB strains with the corresponding 95% Cls in each group, the percent differences (Group 1-Group 3) in the proportion, and the 95% Cls of the differences are presented in Table E for the post-Vaccination 3 evaluable immunogenicity population.

The proportions of subjects achieving a ≥4-fold rise in hSBA titer from baseline to 1 month after Vaccination 3 for the 2 primary MnB test strains were measured for both Group 1 subjects who received MCV4+Tdap+bivalent rLP2086 and Group 3 subjects who received saline+saline+bivalent rLP2086. Of the subjects in Group 1, 84.0% exhibited a ≥4-fold rise in hSBA titer against test strain PMB80 (A22), and 85.7% exhibited a ≥4-fold rise in hSBA titer against test strain PMB2948 (B24). Of the subjects in Group 3, 88.7% exhibited a ≥4-fold rise in hSBA titer against test strain PMB80 (A22) and 87.7% exhibited a ≥4-fold rise in hSBA titer against test strain PMB2948 (B24).

The difference in the proportion of subjects achieving a ≥4-fold rise in hSBA titer between Group 1 and Group 3 at 1 month after Vaccination 3 was −4.7% for PMB80 (A22) (95% Cl: −8.4, −1.1) and −2.1% for PMB2948 (B24) (95% Cl: −5.7, 1.6). The estimated differences of 4-fold response rate were small (<5.0%), with the lower bounds of the 95% CI of the proportion difference being −8.4% (PMB80 [A22]) and −5.7% (PMB2948 [B24]).

TABLE E Comparison of Subjects Achieving hSBA Titer Fold Rise ≥4 From Baseline to 1 Month After Vaccination 3 - Post-Vaccination 3 Evaluable Immunogenicity Population Vaccine Group (as Randomized) Group 1 Group 3 MCV4 + Tdap + rLP2086 Saline + Saline + rLP2086 Difference Strain (Variant) N^(a) n^(b) (%) (95% CI)^(c) N^(a) n^(b) (%) (95% CI)^(c) (%)^(d) (95% CI)^(e) PMB80 (A22) 673 565 (84.0) (81.0, 86.6) 672 596 (88.7) (86.0, 91.0) −4.7 (−8.4, −1.1) PMB2948 (B24) 664 569 (85.7) (82.8, 88.3) 653 573 (87.7) (85.0, 90.2) −2.1 (−5.7, 1.6) Abbreviations: hSBA = serum bactericidal assay using human complement; LLOQ = lower limit of quantitation; LOD = limit of detection. Note: Baseline is defined as the blood draw prior to Vaccination 1. Note: The 4-fold increase is defined as follows: (1) For subjects with a baseline hSBA titer < LOD, or an hSBA titer < LLOQ, a 4-fold response is defined as an hSBA titer ≥4 times the LLOQ. (2) For subjects with a baseline hSBA titer ≥ LLOQ, a 4-fold response is defined as an hSBA titer ≥4 times the baseline titer. ^(a)N = number of subjects with valid and determinate hSBA titers at baseline and at 1 month after Vaccination 3 for the given strain. ^(b)n = Number of subjects achieving at least a 4-fold response at 1 month after Vaccination 3 for the given strain. ^(c)Exact 2-sided confidence interval (CI) based upon the observed proportion of subjects using the Clopper and Pearson method. ^(d)Difference in proportions, expressed as a percentage. ^(e)Exact 2-sided CI and corresponding p-value (based on Chan & Zhang) for the difference in proportions, expressed as a percentage.

Summary of Immune Response to Bivalent rLP2086 and MCV4 and Tdap Antigens When Administered Concomitantly. The noninferiority criteria for bivalent rLP2086 given with MCV4 and Tdap vaccines compared to both MCV4 and Tdap vaccines alone or compared to saline+saline+bivalent rLP2086 required that the lower limits of the 2-sided 95% CI for the GMRs be greater than 0.67 for each of the 10 MCV4/Tdap antigens (1 month after Vaccination 1) and for each of the 2 primary MnB test strains (A22 and B24) (1 month after Vaccination 3). This prespecified threshold was met for both MnB test strains and each of the 10 Tdap and MCV4 antigens.

For the MCV4+Tdap+bivalent rLP2086 group (Group 1) and the MCV4+Tdap+saline group (Group 2), the estimated seroresponse rate differences were between −4.6% and 0.3% for the 6 Tdap and 4 MCV4 antigens, with the lower bounds of the 95% CI of the rate difference equal to 9.1% or less (all differences were greater than −10%). The ≥4-fold rise hSBA responses to 2 primary MnB test strains (A22 and B24) were similar (ranging from 84.0% to 88.7%) between the group that received MCV4+Tdap+bivalent rLP2086 and the group that received saline+saline+bivalent rLP2086.

Immune Response to Bivalent rLP2086. hSBA Titers LLOQ (Descriptive Endpoint).

One of the secondary objectives of this study was to describe the immune response as measured by hSBA performed with 2 primary MnB test strains, 1 expressing a subfamily A LP2086 (PMB80 [A22]) and 1 expressing a subfamily B LP2086 (PMB2948 [B24]), measured 1 month after the second (Visit 4) and the third (Visit 6) vaccinations with bivalent rLP2086.

One of the descriptive secondary endpoints for this secondary objective was the proportion of subjects with hSBA titers ≥LLOQ at 1 month after Vaccination 2 (Visit 4) and at 1 month after Vaccination 3 (Visit 6) to each of the 2 primary MnB test strains. The proportion of subjects with an hSBA titer ≥LLOQ to each of the 2 primary MnB test strains for the post-Vaccination 3 evaluable immunogenicity population is presented in Table F. Note that the LLOQ for PMB80 (A22) was an hSBA titer equal to 1:16, while the LLOQ for PMB2948 (B24) was an hSBA titer equal to 1:8.

For Group 3 (saline+saline+bivalent rLP2086), the proportion of subjects with an hSBA titer ≥LLOQ at baseline (before Vaccination 1) was 5.6% for PMB80 (A22) and 3.4% for PMB2948 (B24). For Group 3 (saline+saline+bivalent rLP2086), the proportions of subjects achieving an hSBA titer ≥LLOQ at 1 month after Vaccination 2 and at 1 month after Vaccination 3 were 68.0% and 91.4%, respectively, for PMB80 (A22) and 66.0% and 92.7%, respectively, for PMB2948 (B24).

For Group 1 (MCV4+Tdap+bivalent rLP2086), the proportion of subjects with an hSBA titer ≥LLOQ at baseline (before Vaccination 1) was 4.4% for PMB80 (A22) and 1.6% for PMB2948 (B24). For Group 1 (MCV4+Tdap+bivalent rLP2086), the proportions of subjects achieving an hSBA titer ≥LLOQ at 1 month after Vaccination 2 and at 1 month after Vaccination 3 were 68.0% and 87.5%, respectively, for PMB80 (A22); and 62.3% and 90.0%, respectively, for PMB2948 (B24).

Compared to before Vaccination 1, a substantial increase in the proportion of subjects with an hSBA titer aLOQ for the 2 primary MnB test strains was observed among both Group 1 and Group 3 subjects at 1 month after Vaccination 2, with additional increases observed at 1 month after Vaccination 3. The hSBA responses in Groups 1 and 3 of the evaluable immunogenicity population were similar.

TABLE F Subjects With hSBA Titer ≥ LLOQ - Post-Vaccination 3 Evaluable Immunogenicity Population Vaccine Group (as Randomized) Group 1 Group 2 Strain (Variant) MCV4 + Tdap + rLP2086 Saline + Saline + rLP2086 Total Sampling Time Point N^(a) n^(b) (%) (95% CI)^(c) N^(a) n^(b) (%) (95% CI)^(c) N^(a) n^(b) (%) (95% CI)^(c) PMB80 (A22) Before Vaccination 1 677 30 (4.4) (3.0, 6.3) 677 38 (5.6) (4.0, 7.6) 1354 68 (5.0) (3.9, 6.3) 1 Month after Vaccination 2 669 455 (68.0) (64.3, 71.5) 665 452 (68.0) (64.3, 71.5) 1334 907 (68.0) (65.4, 70.5) 1 Month after Vaccination 3 679 594 (87.5) (84.8, 89.9) 674 616 (91.4) (89.0, 93.4) 1353 1210 (89.4)  (87.7, 91.0) PMB2948 (B24) Before Vaccination 1 677 11 (1.6) (0.8, 2.9) 676 23 (3.4) (2.2, 5.1) 1353 34 (2.5) (1.7, 3.5) 1 Month after Vaccination 2 656 409 (62.3) (58.5, 66.1) 650 429 (66.0) (62.2, 69.6) 1306 838 (64.2) (61.5, 66.8) 1 Month after Vaccination 3 670 603 (90.0) (87.5, 92.2) 656 608 (92.7) (90.4, 94.6) 1326 1211 (91.3)  (89.7, 92.8) Abbreviations: hSBA = serum bactericidal assay using human complement; LLOQ = lower limit of quantitation. Note: LLOQ = 1:16 for A22; 1:8 for B24. ^(a)N = number of subjects with valid and determinate hSBA titers for the given strain. ^(b)n = Number of subjects with observed hSBA titer ≥ LLOQ for the given strain at the given time point. ^(c)Exact 2-sided confidence interval (CI) based upon the observed proportion of subjects using the Clopper and Pearson method. hSBA Titers Achieving a Defined Level

Another secondary endpoint was the proportion of subjects with hSBA titers ≥LLOQ, ≥4, ≥8, ≥16, ≥32, ≥64, and ≥128 to each of the 2 primary MnB test strains at each blood sampling time point.

Subjects who achieved an hSBA titer ≥4 and ≥16 are described below. An hSBA titer of ≥4 is widely recognized as the correlate of protection against IMD; however, a more conservative hSBA titer of 1:16 has been been considered a level indicative of a 4-fold vaccine effect for subjects seronegative at prevaccination.

The proportions of subjects in Group 1 (MCV4+Tdap+bivalent rLP2086) and Group 3 (saline+saline+bivalent rLP2086) with prevaccination (ie, before Vaccination 1) hSBA titers of ≥4 were 6.9% and 7.5%, respectively, for strain PMB80 (A22); and 2.1% and 3.7%, respectively, for strain PMB2948 (B24). In addition, the proportions of subjects in Groups 1 and 3 with prevaccination hSBA titers of ≥16 were 4.4% and 5.6%, respectively, for strain PMB80 (A22); and 1.5% and 2.1%, respectively, for strain PMB2948 (B24).

In Group 3 (saline+saline+bivalent rLP2086), the proportion of subjects with an hSBA titer ≥4 at 1 month after Vaccination 2 was 68.6% for PMB80 (A22) and 68.2% for PMB2948 (B24). One (1) month after Vaccination 3, the proportion of subjects with an hSBA titer of ≥4 was 91.7% for PMB80 (A22) and 93.1% for PMB2948 (B24). In Group 1 (MCV4+Tdap+bivalent rLP2086), the proportion of subjects with an hSBA titer of ≥4 at 1 month after Vaccination 2 was 68.2% for PMB80 (A22) and 64.8% for PMB2948 (B24). One (1) month after Vaccination 3, the proportion of subjects with an hSBA titer of ≥4 was 87.8% for PMB80 (A22) and 90.7% for PMB2948 (B24).

In Group 3 (saline+saline+bivalent rLP2086), the proportion of subjects with an hSBA titer of ≥16 at 1 month after Vaccination 2 was 68.0% for PMB80 (A22) and 60.3% for PMB2948 (B24). One (1) month after Vaccination 3, the proportion of subjects with an hSBA titer of ≥16 was 91.4% for PMB80 (A22) and 88.9% for PMB2948 (B24). In Group 1 (MCV4+Tdap+bivalent rLP2086), the proportion of subjects with an hSBA titer of ≥16 at 1 month after Vaccination 2 was 68.0% for PMB80 (A22) and 57.6% for PMB2948 (B24). One (1) month after Vaccination 3, the proportion of subjects with an hSBA titer of 1:16 was 87.5% for PMB80 (A22) and 86.7% for PMB2948 (B24).

For both Group 1 and Group 3, the majority of subjects achieved an hSBA titer of 1:16 or greater following 2 or 3 doses of bivalent rLP2086, while relatively few subjects had a measurable hSBA titer to any of the primary MnB test strains at prevaccination Visit 1.

hSBA Geometric Mean Titers. Another secondary endpoint was the hSBA GMTs for each of the 2 primary MnB test strains at each applicable blood sampling time point. Table G presents the hSBA GMTs for each of the 2 primary MnB test strains and the corresponding Cls by sampling time point for Group 1 and Group 3 in the post-Vaccination 3 evaluable immunogenicity population. The GMTs at baseline were below the hSBA LLOQs for both groups. For Group 3 (saline+saline+bivalent rLP2086), hSBA GMTs at 1 month after Vaccination 2 were 23.8 for PMB80 (A22) and 13.0 for PMB2948 (B24). The hSBA GMTs at 1 month after Vaccination 3 were 49.7 for PMB80 (A22) and 27.4 for PMB2948 (B24). For Group 1 (MCV4+Tdap+bivalent rLP2086), hSBA GMTs at 1 month after Vaccination 2 were 23.7 for PMB80 (A22) and 12.0 for PMB2948 (B24). The hSBA GMTs at 1 month after Vaccination 3 were 45.9 for PMB80 (A22) and 24.8 for PMB2948 (B24). The hSBA responses as measured by GMT were similar between Groups 1 and 3.

Reverse Cumulative Distribution Curves for hSBA Titers

The Reverse Cumulative Distribution Curves (RCDCs) showing the distribution of hSBA titers for PMB80 (A22) and PMB2948 (B24) were assessed for Groups 1 and 3 at all sampling time points for the post-Vaccination 3 evaluable immunogenicity population. The RCDCs showed that the majority of subjects responded after Vaccination 2 and had an additional increase in titer for the 2 primary MnB test strains after Vaccination 3. Immune responses to the antigens were similar for Groups 1 and 3.

hSBA ≥4-Fold Response

A prespecified, exploratory objective of this study was to describe the immune response to bivalent rLP2086, as measured by a 4-fold response in hSBA titer performed with 2 primary MnB test strains, 1 expressing LP2086 subfamily A proteins and 1 expressing LP2086 subfamily B proteins, measured at 1 month after the second and third vaccinations (Visit 3 and Visit 5) with bivalent rLP2086.

The endpoints for the exploratory objective were the proportion of subjects achieving at least a 4-fold increase in hSBA titer to 2 MnB strains, PMB80 (A22) and PMB2948 (B24), from baseline to 1 month after the second and third vaccinations with bivalent rLP2086. Note that the LLOQ for PMB80 (A22) was an hSBA titer equal to 1:16, while the LLOQ for PMB80 (A22) was an hSBA titer equal to 1:8. The 4-fold response was defined as follows: For subjects with a baseline hSBA titer below the limit of detection (LOD) or an hSBA titer of <1:4, a 4-fold response was defined as an hSBA titer of ≥16. For subjects with a baseline hSBA titer of ≥LOD (ie, hSBA titer of ≥4) and <LLOQ, a 4-fold response was defined as an hSBA titer ≥4 times the LLOQ. For subjects with a baseline hSBA titer of ≥LLOQ, a 4-fold response was defined as an hSBA titer of times the baseline titer.

The proportion of subjects achieving an hSBA titer fold rise ≥4 for each of the 2 primary MnB test strains for the post-Vaccination 3 evaluable immunogenicity population are presented for Group 1 (MCV4+Tdap+bivalent rLP2086) and Group 3 (saline+saline+bivalent rLP2086) in Table H.

For Group 3 (saline+saline+bivalent rLP2086), the proportion of subjects achieving an hSBA titer fold rise ≥4 from baseline to 1 month after Vaccination 3 was 88.7% for PMB80 (A22) and 87.7% for PMB2948 (B24). At 1 month after Vaccination 2, the proportion of subjects achieving an hSBA titer fold rise ≥4 from baseline was 63.7% for PMB80 (A22) and 58.4% for PMB2948 (B24).

For Group 1 (MCV4+Tdap+bivalent rLP2086), the proportion of subjects achieving an hSBA titer fold rise ≥4 from baseline to 1 month after Vaccination 3 was 84.0% for PMB80 (A22) and 85.7% for PMB2948 (B24). At 1 month after Vaccination 2, the proportion of subjects achieving an hSBA titer fold rise ≥4 from baseline was 64.3% for PMB80 (A22) and 56.3% for PMB2948 (B24).

The hSBA responses as measured by 4-fold titer response were similar between Groups 1 and 3.

TABLE G hSBA Geometric Mean Titers - Post-Vaccination 3 Evaluable Immunogenicity Population Vaccine Group (as Randomized) Group 1 Group 3 Strain (Variant) MCV4 + Tdap + rLP2086 Saline + Saline + rLP2086 Total Sampling Time Point N^(a) GMT^(b) (95% CI)^(c) N^(a) GMT^(b) (95% CI)^(c) N^(a) GMT^(b) (95% CI)^(c) PMB80 (A22) Before Vaccination 1 677 8.5 (8.28, 8.64) 677 8.6 (8.39, 8.84) 1354 8.5 (8.39, 8.68) 1 Month after Vaccination 2 669 23.7 (22.10, 25.40) 665 23.8 (22.14, 25.54) 1334 23.7 (22.58, 24.95) 1 Month after Vaccination 3 679 45.9 (42.74, 49.35) 674 49.7 (46.43, 53.30) 1353 47.8 (45.47, 50.23) PMB2948 (B24) Before Vaccination 1 677 4.1 (4.04, 4.19) 676 4.2 (4.10, 4.27) 1353 4.2 (4.09, 4.21) 1 Month after Vaccination 2 656 12.0 (11.12, 12.99) 650 13.0 (12.03, 14.13) 1306 12.5 (11.83, 13.23) 1 Month after Vaccination 3 670 24.8 (23.11, 26.58) 656 27.4 (25.58, 29.41) 1326 26.1 (24.80, 27.38) Abbreviations: GMT = geometric mean titer; hSBA = serum bactericidal assay using human complement; LLOQ = lower limit of quantitation. Note: LLOQ = 1:16 for A22; 1:8 for B24. Titers below the LLOQ were set to 0.5 × LLOQ for analysis. ^(a)N = number of subjects with valid and determinate hSBA titers for the given strain. ^(b)GMTs were calculated using all subjects with valid and determinate hSBA titers at the given time point. ^(c)Confidence intervals (CIs) are back transformations of confidence levels based on the Student t distribution for the mean logarithm of assay results.

TABLE H Subjects Achieving ≥4-Fold Rise in hSBA Titer - Post-Vaccination 3 Evaluable Immunogenicity Population Vaccine Group (as Randomized) Group 1 Group 3 Strain (Variant) MCV4 + Tdap + rLP2086 Saline + Saline + rLP2086 Total Sampling Time Point N^(a) n^(b) (%) (95% CI)^(c) N^(a) n^(b) (%) (95% CI)^(c) N^(a) n^(b) (%) (95% CI)^(c) hSBA Titer fold rise ≥4 from baseline PMB80 (A22) 1 Month after Vaccination 2 663 426 (64.3) (60.5, 67.9) 663 422 (63.7) (59.9, 67.3) 1326  848 (64.0) (61.3, 66.5) 1 Month after Vaccination 3 673 565 (84.0) (81.0, 86.6) 672 596 (88.7) (86.0, 91.0) 1345 1161 (86.3) (84.4, 88.1) PMB2948 (B24) 1 Month after Vaccination 2 650 366 (56.3) (52.4, 60.2) 647 378 (58.4) (54.5, 62.3) 1297  744 (57.4) (54.6, 60.1) 1 Month after Vaccination 3 664 569 (85.7) (82.8, 88.3) 653 573 (87.7) (85.0, 90.2) 1317 1142 (86.7) (84.8, 88.5) Abbreviations: hSBA = serum bactericidal assay using human complement; LLOQ = lower limit of quantitation; LOD = limit of detection. Note: LLOQ = 1:16 for A22; 1:8 for B44. Note: Baseline is defined as the blood draw prior to Vaccination 1. Note: The 4-fold increase is defined as follows: (1) For subjects with a baseline hSBA titer < LOD (hSBA titer <1:4), a response is defined as an hSBA titer ≥1:16. (2) For subjects with a baseline hSBA titer ≥ LOD (hSBA titer ≥1:4) and < LLOQ, a response is defined as an hSBA titer ≥4 times the LLOQ. (3) For subjects with a baseline hSBA titer ≥ LLOQ, a 4-fold response is defined as an hSBA titer ≥4 times the baseline titer. ^(a)N = number of subjects with valid and determinate hSBA titers for the given strain at both the specified time point and baseline. ^(b)n = number of subjects who achieved hSBA titer fold rise ≥4 from baseline for the given strain. ^(c)Exact 2-sided confidence interval (CI) based upon the observed proportion of subjects using the Clopper and Pearson method. Additional hSBA Fold Response

An additional endpoint was the proportion of subjects achieving at least a 2-fold increase and the proportion of subjects achieving at least a 3-fold increase in hSBA titer from baseline at each postvaccination blood sampling visit for each of the 2 primary MnB strains. Note that the LLOQ for PMB80 (A22) was an hSBA titer equal to 1:16, while the LLOQ for PMB2948 (B24) was an hSBA titer equal to 1:8.

The proportions of subjects achieving a 2-fold rise in hSBA titer from baseline to 1 month after Vaccination 2 for Group 1 and Group 3 for MnB strains were 66.2% and 66.1%, respectively, for PMB80 (A22); and 56.8% and 59.7%, respectively, for PMB2948 (B24). The proportions of subjects achieving an hSBA titer fold rise ≥2 from baseline to 1 month after Vaccination 3 for Group 1 and Group 3 for MnB strains were 86.6% and 90.3%, respectively, for PMB80 (A22); and 86.1% and 88.5%, respectively, for PMB2948 (B24).

The proportions of subjects achieving a fold rise in hSBA titer from baseline to 1 month after Vaccination 2 for Group 1 and Group 3 for MnB strains were 64.3% and 63.7%, respectively, for PMB80 (A22); and 56.3% and 58.4%, respectively, for PMB2948 (B24). The proportions of subjects achieving a fold rise in hSBA titer from baseline to 1 month after Vaccination 3 for Group 1 and Group 3 for MnB strains were 84.0% and 88.7%, respectively, for PMB80 (A22); and 85.7% and 87.7%, respectively, for PMB2948 (B24).

For subjects with baseline titer below LOD (1:4) in the post-Vaccination 3 evaluable immunogenicity population, the majority of subjects achieved a 2-fold, 3-fold, and 4-fold response in both Group 1 and Group 3 at all blood sampling time points, ranging from 56.7% to 66.6% 1 month after Vaccination 2, and from 86.3% to 91.0% 1 month after Vaccination 3.

Summary of Immune Response to Bivalent rLP2086

In summary of the descriptive endpoints under the secondary objectives, the majority of subjects achieved an hSBA titer ≥LLOQ for both Group 1 (MCV4+Tdap+bivalent rLP2086) and Group 3 (saline+saline+bivalent rLP2086) to both primary MnB test strains, while only a very small proportion of subjects had measurable hSBA titers ≥LLOQ at baseline (prevaccination Visit 1). Substantial immune responses with the 2 MnB strains were observed at 1 month after Vaccination 2, with additional increases observed at 1 month after Vaccination 3 for both Group 1 and Group 3 subjects. This conclusion was confirmed by the proportion of subjects with an hSBA titer of 1:16 following 3 doses, the observed GMTs achieved after 2 doses and after 3 doses in both groups, and the RCDCs for the 2 primary MnB test strains.

For both Group 1 (MCV4+Tdap+bivalent rLP2086) and Group 3 (saline+saline+bivalent rLP2086), a high proportion of subjects achieved an hSBA titer fold rise ≥4 for each of the 2 primary MnB test strains.

In addition, the majorities of subjects achieved an hSBA titer fold rise ≥2 and an hSBA titer fold rise ≥3 for the 2 primary MnB strains at all sampling time points for both Group 1 (MCV4+Tdap+bivalent rLP2086) and Group 3 (saline+saline+bivalent rLP2086). The proportion of subjects with results meeting these criteria was higher after 3 vaccinations compared to 2 vaccinations. Sensitivity analyses also support the robust immune responses induced by 3 doses for both groups.

These results support the evidence that the immune response to bivalent rLP2086 when coadministered with the MCV4+Tdap vaccines yields a robust immune response that is comparable to the immune response to bivalent rLP2086+saline.

Immune Response to MCV4 and Tdap

Tdap/MCV4 Antigen GMTs and GMCs

Table I presents the GMTs and GMCs and the corresponding Cls for each of the Tdap and MCV4 antigens at baseline and 1 month after Vaccination 1 for Group 1 (MCV4+Tdap+bivalent rLP2086) and Group 2 (MCV4+Tdap+saline) in the post-Vaccination 1 evaluable immunogenicity population.

For Group 2 (MCV4+Tdap+saline), the Tdap antigen GMCs at baseline (before Vaccination 1) and at 1 month after Vaccination 1 were 0.2 and 9.8, respectively (diphtheria toxoid); 0.5 and 10.3, respectively (tetanus toxoid); 3.9 and 14.2, respectively (pertussis toxoid); 21.8 and 122.9, respectively (pertussis filamentous hemagglutinin); 16.2 and 228.9, respectively (pertussis pertactin); 8.5 and 154.2, respectively (pertussis fimbrial agglutinogens types 2+3); and the MCV4 antigen GMTs at baseline (before Vaccination 1) and at 1 month after Vaccination 1 were 436.3 and 5113.0, respectively (serogroup A); 84.3 and 1650.2, respectively (serogroup C); 168.5 and 2244.9, respectively (serogroup Y); and 177.7 and 6367.9, respectively (serogroup W-135). For Group 1 (MCV4+Tdap+bivalent rLP2086), the Tdap antigen GMs at baseline (before Vaccination 1) and at 1 month after Vaccination 1 were 0.2 and 9.3, respectively (diphtheria toxoid); 0.5 and 9.4, respectively (tetanus toxoid); 3.9 and 13.2, respectively (pertussis toxoid); 21.3 and 112.0, respectively (pertussis filamentous hemagglutinin); 15.7 and 202.0, respectively (pertussis pertactin); 8.4 and 138.1, respectively (pertussis fimbrial agglutinogens types 2+3); and the MCV4 antigen GMs at baseline (before Vaccination 1) and at 1 month after Vaccination 1 were 490.0 and 4647.3, respectively (serogroup A); 87.6 and 1679.2, respectively (serogroup C); 183.3 and 2212.6, respectively (serogroup Y); and 176.7 and 5925.1, respectively (serogroup W-135). Overall, the estimated GMs were similar between Group 1 and Group 2.

IgG MCV4 GMCs

An exploratory objective of this study was to describe the IgG immune response as measured by anti-MCV4 IgG GMCs induced by the MCV4 vaccine when given with and without bivalent rLP2086 as measured by anti-MCV IgG GMCs. Exploratory immunogenicity endpoints for this study were applied to the 4 MCV4 antigenic components in subjects from Group 1 (MCV4+Tdap+bivalent rLP2086) and Group 2 (MCV4+Tdap+saline). The exploratory endpoints were the IgG response (measured as GMCs) to serogroup A, C, Y or W-135 plysaccharide. Following the validation of this assay (ie, postvalidation) and prior to sample testing, the LLOQs were adjusted upwards based on additional data analyses from standard reference curves to: 0.1795 μg/mL for serogroup A, 0.0940 μg/mL for serogroup C, 0.2485 μg/mL for serogroup Y, and 0.2530 μg/mL for serogroup W-135. Results below the LLOQ were set to 0.5×LLOQ for analysis. MCV4 IgG GMCs for Group 1 and Group 2 in the post-Vaccination 1 evaluable immunogenicity population using the adjusted postvalidation LLOQs are presented in Table J.

For Group 1 (MCV4+Tdap+rLP2086), the MCV4 IgG GMCs at baseline (before Vaccination 1) and at 1 month after Vaccination 1 were: 0.17 and 11.42 μg/mL, respectively (serogroup A); 0.11 and 5.59 μg/m L, respectively (serogroup C); 0.14 and 2.49 μg/mL, respectively (serogroup Y); and 0.13 and 1.79 μg/m L, respectively (serogroup W-135). For Group 2 (MCV4+Tdap+saline), the MCV4 IgG GMCs at baseline (before Vaccination 1) and at 1 month after Vaccination 1 were: 0.15 and 11.38 μg/m L, respectively (serogroup A); 0.11 and 5.47 μg/m L, respectively (serogroup C); 0.13 and 2.14 μg/mL, respectively (serogroup Y); and 0.13 and 1.84 μg/mL, respectively (serogroup W-135).

Overall, the MCV4 IgG GMCs were numerically similar for Group 1 and Group 2. Based on the initial assay validation data analyses, the MCV4 IgG assay LLOQs were set at 0.1037 μg/mL for serogroup A, 0.0204 μg/mL for serogroup C, 0.1104 μg/mL for serogroup Y, and 0.1198 μg/mL for serogroup W-135. MCV4 IgG GMCs for Group 1 and Group 2 in the post-Vaccination 1 evaluable immunogenicity population using the validation LLOQs also showed that the MCV4 IgG GMCS were numerically similar for Group 1 and Group 2.

Subjects Achieving Prespecified Antibody Level for Diphtheria Toxoid/Tetanus Antigens

A descriptive secondary endpoint of the study was the proportion of subjects (Groups 1 and 2) who achieve an antibody level 1.0 IU/mL to tetanus toxoid and proportion of subjects (Groups 1 and 2) who achieve an antibody level 1.0 IU/mL to diphtheria toxoid.

The proportion of subjects achieving the prespecified level of antibodies to diphtheria toxoid and tetanus toxoid antigens, with corresponding 95% CI are presented in Table K for Group 1 (MCV4+Tdap+bivalent rLP2086) and Group 2 (MCV4+Tdap+saline) in the post-Vaccination 1 evaluable immunogenicity population.

For Group 1 (MCV4+Tdap+bivalent rLP2086), the proportion of subjects achieving the prespecified level of antibodies to diphtheria toxoid and tetanus toxoid antigens 1 month after Vaccination 1 was 98.1% (95% Cl: 96.8, 98.9) and 99.1% (95% Cl: 98.2, 99.6), respectively.

For Group 2 (MCV4+Tdap+saline), the proportion of subjects achieving the prespecified level of antibodies to diphtheria toxoid and tetanus antigens 1 month after Vaccination 1 was 99.0% (95% Cl: 98.0, 99.6) and 99.0% (95% Cl: 98.0, 99.6), respectively.

TABLE I Tdap/MCV4 Antigen Geometric Means - Post-Vaccination 1 Evaluable Immunogenicity Population Vaccine Group (as Randomized) Tdap/MCV4 Group 1 Group 2 Antigen MCV4 + Tdap + rLP2086 MCV4 + Tdap + Saline Total Sampling Time Point N^(a) GM^(b) (95% CI)^(c) N^(a) GM^(b) (95% CI)^(c) N^(a) GM^(b) (95% CI)^(c) Tdap Diphtheria Before Vaccination 1 775 0.2 (0.15, 0.18) 781 0.2 (0.15, 0.17) 1556 0.2 (0.15, 0.17) 1 Month after Vaccination 1 778 9.3 (8.67, 9.92) 780 9.8  (9.23, 10.51) 1558 9.6  (9.12, 10.02) Tetanus Before Vaccination 1 775 0.5 (0.43, 0.50) 781 0.5 (0.44, 0.52) 1556 0.5 (0.44, 0.50) 1 Month after Vaccination 1 778 9.4 (8.95, 9.98) 780 10.3  (9.75, 10.85) 1558 9.9  (9.49, 10.24) Pertussis toxoid Before Vaccination 1 775 3.9 (3.48, 4.26) 781 3.9 (3.54, 4.29) 1556 3.9 (3.62, 4.15) 1 Month after Vaccination 1 778 13.2 (12.35, 14.14) 780 14.2 (13.28, 15.20) 1558 13.7 (13.06, 14.37) Pertussis filamentous hemagglutinin Before Vaccination 1 775 21.3 (19.66, 23.12) 781 21.8 (20.11, 23.62) 1556 21.6 (20.36, 22.82) 1 Month after Vaccination 1 778 112.0 (106.15, 118.14) 780 122.9 (116.42, 129.84) 1558 117.3 (112.94, 121.92) Pertussis pertactin Before Vaccination 1 775 15.7 (14.29, 17.30) 781 16.2 (14.81, 17.66) 1556 15.9 (14.95, 17.02) 1 Month after Vaccination 1 778 202.0 (187.77, 217.25) 780 228.9 (212.72, 246.35) 1558 215.0 (204.20, 226.47) Pertussis fimbriae agglutinogens types 2 + 3 Before Vaccination 1 775 8.4 (7.88, 9.02) 781 8.5 (7.94, 9.14) 1556 8.5 (8.07, 8.90) 1 Month after Vaccination 1 778 138.1 (121.20, 157.33) 780 154.2 (135.30, 175.79) 1558 145.9 (133.07, 160.06) rSBA MCV4 Serogroup A Before Vaccination 1 726 490.0 (450.15, 533.38) 745 436.3 (401.66, 473.89) 1471 462.0 (435.45, 490.20) 1 Month after Vaccination 1 763 4647.3 (4317.66, 772 5113.0 (4748.73, 1535 4875.9 (4628.23, 5136.92) 5002.09) 5505.17) Serogroup C Before Vaccination 1 748 87.6 (79.78, 96.23) 756 84.3 (77.00, 92.38) 1504 86.0 (80.53, 91.75) 1 Month after Vaccination 1 768 1679.2 (1539.63, 767 1650.2 (1519.01, 1535 1664.6 (1567.82, 1767.39) 1831.38) 1792.65) Serogroup Y Before Vaccination 1 762 183.3 (170.25, 197.43) 764 168.5 (157.41, 180.37) 1526 175.8 (167.13, 184.82) 1 Month after Vaccination 1 771 2212.6 (2056.08, 770 2244.9 (2088.70, 1541 2228.7 (2117.07, 2346.26) 2381.08) 2412.89) Serogroup W-135 Before Vaccination 1 755 176.7 (161.93, 192.74) 767 177.7 (163.47, 193.17) 1522 177.2 (166.83, 188.19) 1 Month after Vaccination 1 751 5925.1 (5469.77, 765 6367.9 (5872.68, 1516 6144.6 (5804.87, 6504.11) 6418.33) 6904.88) Abbreviations: ELISA = enzyme-linked immunosorbent assay; EU = ELISA units; GM = geometric mean; LLOQ = lower limit of quantitation; MCV4 = quadrivalent meningococcal polysaccharide conjugate (vaccine); rSBA = serum bactericidal assay using rabbit complement; Tdap = tetanus, diphtheria, and acellular pertussis (vaccine). Note: LLOQ = 0.037 IU/mL for diphtheria; 0.05 IU/mL for tetanus; 0.9 EU/mL for pertussis toxoid; 2.9 EU/mL for pertussis filamentous hemagglutinin; 3.0 EU/mL for pertussis pertactin; 10.6 EU/mL for pertussis fimbriae agglutinogens types 2 + 3. 1:32 for rSBA serogroup A, 1:64 for serogroup C, 1:128 for serogroup Y, and 1:64 for serogroup W-135. Results below the LLOQ were set to 0.5 × LLOQ for analysis. ^(a)N = number of subjects with valid and determinate assay results for the given antigen. ^(b)GMs were calculated using all subjects with valid and determinate assay results. ^(c)Confidence intervals (CIs) are back transformations of confidence levels based on the Student t distribution for the mean logarithm of assay results.

TABLE J IgG MCV4 Geometric Mean Concentrations (Postvalidation LLOQs) - Post-Vaccination 1 Evaluable Immunogenicity Population Vaccine Group (as Randomized) Group 1 Group 2 Antigen MCV4 + Tdap + rLP2086 MCV4 + Tdap + Saline Total Sampling Time Point N^(a) GMC^(b) (95% CI)^(c) N^(a) GMC^(b) (95% CI)^(c) N^(a) GMC^(b) (95% CI)^(c) Serogroup A Before Vaccination 1 779 0.17 (0.16, 0.19) 781 0.15 (0.14, 0.17) 1560 0.16 (0.15, 0.17) 1 Month after Vaccination 1 779 11.42 (10.30, 12.65) 781 11.38 (10.21, 12.67) 1560 11.40 (10.58, 12.28) Serogroup C Before Vaccination 1 779 0.11 (0.10, 0.12) 781 0.11 (0.10, 0.12) 1560 0.11 (0.10, 0.12) 1 Month after Vaccination 1 779 5.59 (4.90, 6.39) 781 5.47 (4.79, 6.23) 1560 5.53 (5.04, 6.07) Serogroup Y Before Vaccination 1 779 0.14 (0.13, 0.14) 781 0.13 (0.13, 0.14) 1560 0.14 (0.13, 0.14) 1 Month after Vaccination 1 779 2.49 (2.22, 2.79) 781 2.14 (1.92, 2.39) 1560 2.31 (2.13, 2.50) Serogroup W-135 Before Vaccination 1 779 0.13 (0.13, 0.14) 781 0.13 (0.13, 0.14) 1560 0.13 (0.13, 0.14) 1 Month after Vaccination 1 779 1.79 (1.59, 2.01) 781 1.84 (1.62, 2.09) 1560 1.81 (1.66, 1.98) Abbreviations: GMC = geometric mean concentration; IgG = immunoglobulin G; LLOQ = lower limit of quantitation. Note: LLOQ = 0.1795 μg/mL for IgG MCV4 serogroup A, 0.0940 μg/mL for IgG MCV4 serogroup C, 0.2485 μg/mL for IgG MCV4 serogroup Y, and 0.2530 μg/mL for IgG MCV4 serogroup W-135. Results below the LLOQ were set to 0.5 × LLOQ for analysis. ^(a)N = number of subjects with valid and determinate assay results for the given serogroup. ^(b)GMCs were calculated using all subjects with valid and determinate assay results. ^(c)Confidence intervals (CIs) are back transformations of confidence levels based on the Student t distribution for the mean logarithm of assay results.

TABLE K Subjects Achieving Prespecified Antibody Level for Diphtheria Toxoid/Tetanus Antigens - Post- Vaccination 1 Evaluable Immunogenicity Population Vaccine Group (as Randomized) Antigen Group 1 Group 2 Sampling Antibody MCV4 + Tdap + rLP2086 MCV4 + Tdap + Saline Total Time Point Level N^(a) n^(b) (%) (95% CI)^(c) N^(a) n^(b) (%) (95% CI)^(c) N^(a) n^(b) (%) (95% CI)^(c) Diphtheria toxoid Before ≥1.0 IU/mL 775 32 (4.1) (2.8, 5.8) 781 35 (4.5) (3.1, 6.2) 1556  67 (4.3) (3.4, 5.4) Vaccination 1 1 Month after ≥1.0 IU/mL 778 763 (98.1) (96.8, 98.9) 780 772 (99.0) (98.0, 99.6) 1558 1535 (98.5) (97.8, 99.1) Vaccination 1 Tetanus Before ≥1.0 IU/mL 775 202 (26.1) (23.0, 29.3) 781 205 (26.2) (23.2, 29.5) 1556  407 (26.2) (24.0, 28.4) Vaccination 1 1 Month after ≥1.0 IU/mL 778 771 (99.1) (98.2, 99.6) 780 772 (99.0) (98.0, 99.6) 1558 1543 (99.0) (98.4, 99.5) Vaccination 1 ^(a)N = number of subjects with valid and determinate assay results for the given antigen. ^(b)n = Number of subjects with antibody concentration ≥ prespecified level for the given antigen. ^(c)Exact 2-sided confidence intervals (CIs) based upon the observed proportion of subjects using the Clopper and Pearson method. RCDCs for MCV4 and Tdap Antigens

The RCDCs showing the distribution of rSBA titers for MCV4 serogroups A, C, Y, and W-135 for Group 1 (MCV4+Tdap+bivalent rLP2086) and Group 2 (MCV4+Tdap+saline), before and 1 month after Vaccination 1, for the post-Vaccination 1 evaluable immunogenicity population showed robust immune responses among subjects after Vaccination 1 for both groups and the curves were similar throughout the range of rSBA titers.

The RCDCs showing the distribution of IgG titers to MCV4 serogroup A, C, Y, and W-135 polysaccharides using the postvalidation LLOQs for Group 1 (MCV4+Tdap+bivalent rLP2086) and Group 2 (MCV4+Tdap+saline), before and 1 month after Vaccination 1, for the post-Vaccination 1 evaluable immunogenicity population, and the RCDCs showing the distribution of IgG titers for MCV4 serogroups A, C, Y, and W-135 using the validation LLOQs for Group 1 (MCV4+Tdap+bivalent rLP2086) and Group 2 (MCV4+Tdap+saline), before and 1 month after Vaccination 1, for the post-Vaccination 1 evaluable immunogenicity population showed robust immune responses among subjects after Vaccination 1 for both groups using both the validation and postvalidation LLOQs.

The RCDCs showing the distribution of Tdap titers for diphtheria, tetanus, pertussis toxoid, pertussis filamentous hemagglutinin, pertussis pertactin, and pertussis fimbrial agglutinogens types 2+3 for Group 1 (MCV4+Tdap+bivalent rLP2086) and Group 2 (MCV4+Tdap+saline), before and 1 month after Vaccination 1, for the post-Vaccination 1 evaluable immunogenicity population showed robust immune responses among subjects after Vaccination 1 for both groups.

Immune Response to MCV4 and Tdap

The GMs of antibodies to Tdap/MCV4 antigens were similar between Group 2 (MCV4+Tdap+saline) and Group 1 (MCV4+Tdap+bivalent rLP2086), and the observed Tdap/MCV4 antigen GMs after Vaccination 1 were indicative of a robust immune response for both groups. RCDCs also supported robust immune responses after Vaccination 1 for both Group 1 and Group 2.

The noninferiority criteria for MCV4+Tdap+bivalent rLP2086 compared to bivalent rLP2086+saline+saline and MCV4+Tdap+saline required that the lower limit of the 2-sided 95% Cls for the GMRs were greater than 0.67 for each of the 10 MCV4/Tdap antigens (1 month after Vaccination 1) and for each of the hSBA titers using 2 primary MnB test strains (PMB80 [A22] and PMB2948 [B24]) (1 month after Vaccination 3). This prespecified threshold was met for both MnB test strains and all 10 Tdap and MCV4 antigens. The coprimary immunogenicity objectives of the study were met as they satisfied the pre-specified non-inferiority criteria.

The immune response induced by MCV4 vaccine and Tdap vaccines given concomitantly with bivalent rLP2086 was noninferior to the immune response induced by MCV4 and Tdap vaccines alone when measured 1 month after the first vaccination in both groups. The immune response to all antigenic components of the MCV4 and Tdap vaccines were assessed.

The immune response induced by bivalent rLP2086 given concomitantly with MCV4 and Tdap vaccines, as measured by hSBA performed with 2 MnB test strains was noninferior to the immune response induced by bivalent rLP2086 alone, when measured 1 month after the third vaccination with bivalent rLP2086 in both groups.

For the MCV4+Tdap+bivalent rLP2086 group (Group 1) and the MCV4+Tdap+saline group (Group 2), the estimated differences of the seroresponse rates were between −4.6% and 0.3% for the 6 Tdap and 4 MCV4 antigens, with the lower bounds of the 95% CI of the rate difference equal to 9.1% or less. Since the Cls of the differences were all greater than −10%; these differences were not clinically significant. The RCDCs showed robust immune responses after Vaccination 1 for subjects in both groups. Robust GMTs relative to baseline were observed for both groups that received MCV4+Tdap.

The 4-fold rise responses to the 2 primary MnB test strains PMB80 (A22) and PMB2958 (B24) from baseline to 1 month after Vaccination 3 were similar between the group that received MCV4+Tdap+bivalent rLP2086 (84.0% and 85.7%, respectively) and the group that received bivalent rLP2086+saline+saline (88.7% and 87.7%, respectively).

Immunogenicity Discussion

The noninferiority criteria for MCV4+Tdap+bivalent rLP2086 compared to bivalent rLP2086+saline+saline and MCV4+Tdap+saline required that the lower limit of the 2-sided 95% Cls for the GMRs was greater than 0.67 (ie, at a 1.5 noninferiority margin) for each of the 10 MCV4/Tdap antigens (1 month after Vaccination 1) and for each of the hSBA titers using 2 primary MnB test strains (A22 and B24) (1 month after Vaccination 3). This prespecified threshold was met for both MnB test strains and the 10 Tdap and MCV4 antigens. Thus, both coprimary immunogenicity objectives were met. Specifically, these data support at least the following: 1) The immune response (based on GMs) induced by MCV4 and Tdap vaccines given with bivalent rLP2086 was noninferior to the immune response induced by MCV4 and Tdap vaccines alone, when measured 1 month after the first vaccination in both groups. The immune response to all components of MCV4 and Tdap vaccines were assessed. 2) The immune response (based on GMT) induced by bivalent rLP2086, as measured by hSBA performed with 2 MnB test strains, given with MCV4 and Tdap vaccines, was noninferior to the immune response induced by bivalent rLP2086 alone, when measured 1 month after the third vaccination with bivalent rLP2086 in both groups.

In this randomized, observer-blinded, active-controlled multicenter trial in subjects aged ≥10 to <13 years, the immunogenicity for both bivalent rLP2086 and MCV4+Tdap vaccines remained robust when vaccines were administered concomitantly.

Overall, these data indicate that Tdap, MCV4, MenACWY-CRM, and HPV vaccines may be given concomitantly clinically and these vaccines are recommended to be given at a single visit. The data from this current study (B1971015) supports that conclusion and also that bivalent rLP2086 may be given with Tdap and MCV4.

This study showed that at least 2 doses of bivalent rLP2086 elicited a robust immune response to both primary MnB test strains. This study also showed that 3 doses of bivalent rLP2086 elicited a robust immune response to both primary MnB test strains (A22 and B24). The fHBP expressed on these test strains are heterologous to rLP2086 vaccine antigens and represent the most prevalent subfamily A and subfamily B variants identified among invasive meningococcal disease isolates in the USA. These 2 variants also are prominent among disease isolates in other geographic regions. The immunogenicity assessments for study B1971015 employed definitions of 4-fold increases in hSBA titers.

The LOD for each of the 2 primary MnB test strains was a titer equal to 1:4 (recognized as the correlate of protection), and the validation LLOQs for these 2 primary MnB test strains were hSBA titers equal to 1:8 for PMB2948 (B24) and 1:16 for PMB80 (A22). Prior to receipt of bivalent rLP2086, a low proportion of study subjects had hSBA titers indicating that most subjects in this population could be considered unprotected against MnB disease. After the second study vaccination, a high proportion of subjects achieved 4-fold increases in hSBA titers and exhibited post-immunization titers 1:4 and LLOQ. These response rates were similar between those subjects receiving coadministration of bivalent rLP2086 with MCV4+Tdap and those receiving bivalent rLP2086 with saline for both MnB strains. The RCDCs also showed that the majority of subjects elicited responses to the 2 primary MnB strains, irrespective of whether bivalent rLP2086 was coadministered with MCV4+Tdap.

In the current study (B1971015), the 4-fold response rates (95% CI) after the second and third dose of bivalent rLP2086 for PMB80 (A22) when Group 1 and Group 2 were combined was 64.0% (61.3, 66.5) and 86.3% (84.4, 88.1). Similarly for B24 the 4-fold response rates (95% Cl) after the second and third dose of bivalent rLP2086 for Group 1 and Group 2 combined was 57.4% (54.6, 60.1) and 86.7% (84.8, 88.5).

Of note, the immune responses after the second dose of rLP2086 in study B1971015 were robust as assessed by both the 4-fold increases in hSBA titers from baseline and by the proportions of subjects with post-vaccination hSBA titers ≥LLOQ (1:16 for 22 and 1:8 for B24). For A22 the percentage (95% CI) of subjects in Groups 1 and 2, combined, who achieved an hSBA titer ≥LLOQ (1:16) after the second dose was 68.0% (65.4, 70.5). For B24, 64.2% (61.5, 66.8) of subjects achieved an hSBA titer LLOQ (1:8) after second study dose. These data show substantial hSBA responses to 2 doses of bivalent rLP2086.

Overall, these results indicate that the immune response to MCV4+Tdap and the immune response to bivalent rLP2086 remain robust when bivalent rLP2086 and MCV4+Tdap are administered concomitantly and satisfied the coprimary study objectives.

These data in subjects aged ≥10 to <13 years satisfied the coprimary study objectives, supporting the thesis that bivalent rLP2086 can be given concomitantly with MCV4+Tdap. Moreover, high proportions of subjects achieved hSBA 4-fold responses and hSBA titers ≥LLOQ after vaccination with 2 or 3 doses of bivalent rLP2086 and these robust responses were not impacted by concomitant use of MCV4+Tdap. When considering the immunogenicity and the safety data together, the benefit-risk profile supports the administration of bivalent rLP2086 either alone or with MCV4+Tdap.

Example 12: A Phase 3, Randomized, Active Controlled, Observer Blinded Trial to Assess the Safety and Tolerability of a Meningococcal Serogroup B Bivalent Recombinant Lipoprotein (rLP2086) Vaccine Given in Healthy Subjects Aged ≥10 to <26 Years

This was a Phase 3, randomized, active-controlled, observer-blinded, multicenter trial designed to assess the safety and tolerability of bivalent rLP2086 in healthy subjects aged ≥10 to <26 years. Approximately 5,700 subjects were to be randomly assigned to 1 of 2 groups in a 2:1 ratio. Group 1 received bivalent rLP2086 at Month 0 (Day 1) followed by subsequent vaccinations at Months 2 and 6. Group 2 received a hepatitis A virus (HAV) vaccine at Month 0 and Month 6 and a saline injection at Month 2.

The Bivalent rLP2086 vaccine was in a 0.5 mL dose formulated to contain 60 μg each of a purified subfamily A rLP2086 protein (variant A05, SEQ ID NO: 1) and a purified subfamily B rLP2086 protein (variant B01, SEQ ID NO: 2), 0.15 M sodium chloride, 2.8 molar ratio polysorbate 80, and 0.25 mg of Al³⁺ as aluminum phosphate (AIPO₄) in 10 mM histidine-buffered saline at pH 6.0.

The HAV vaccine (HAVRIX, 0.5-mL dose or 1.0-mL dose) contained 720 ELISA units (EL.U.) or 1440 EL.U. of viral antigen. In particular, HAVRIX (Hepatitis A Vaccine) is a sterile suspension of inactivated virus for intramuscular administration. The virus (strain HM175) is propagated in MRC-5 human diploid cells. Age-specific doses of HAV vaccine (HAVRIX) were supplied according to country-specific guidelines. Each 1-mL adult dose of vaccine contains 1440 EL.U. of viral antigen, adsorbed on 0.5 mg of aluminum as aluminum hydroxide. Each 0.5-mL pediatric dose of vaccine contains 720 EL.U. of viral antigen, adsorbed onto 0.25 mg of aluminum as aluminum hydroxide. HAVRIX contains the following excipients: Amino acid supplement (0.3% w/v) in a phosphate-buffered saline solution and polysorbate 20 (0.05 mg/mL). From the manufacturing process, HAVRIX also contains residual MRC-5 cellular proteins (not more than 5 mcg/mL), formalin (not more than 0.1 mg/mL), and neomycin sulfate (not more than 40 ng/mL), an aminoglycoside antibiotic included in the cell growth media. HAVRIX is formulated without preservatives.

Sterile normal saline solution (0.9% sodium chloride) for injection, was supplied as a 0.5-mL dose.

TABLE A Study Design Final Telephone Telephone Telephone Telephone Follow-up Telephone Vax 1 Contact Vax 2 Contact Contact Contact Vax 3 Visit Contact Visit 1 2 3 4 5 6 7 8 9 Number Approx 0 1 2 3 4 5 6 7 12 month Group 1 rLP2086 rLP2086 rLP2086 (N = 3800) Group 2 HAV Normal HAV (N = 1900) vaccine saline vaccine Abbreviations: approx = approximate; Vax = vaccinnation. This study assessed the safety and tolerability of bivalent rLP2086 at the 120-μg dose level administered in healthy subjects aged ≥10 to <26 years using a 0-, 2-, and 6-month schedule and provided an important component of the safety database for bivalent rLP2086.

HAV vaccine administered at Months 0 and 6 was chosen as the control in this study because there was no proven safe, immunogenic, and effective vaccine against MnB that could serve as an active control. In comparison to other recommended vaccines for this age group, HAV vaccine has a well-established tolerability profile. In addition, HAV vaccine conferred a benefit to subjects who may become at increased risk for hepatitis A viral infection either during future travel or other exposures. Saline was administered at Month 2 in the control group to maintain the study blind. Subjects were ineligible to participate if they met any of the exclusion criteria, including: Previous vaccination with any meningococcal serogroup B vaccine; Subjects who had received prior HAV vaccination; Contraindication to vaccination with any HAV vaccine. Subjects received 1 dose of 120 μg of bivalent rLP2086 or HAV vaccine/saline/HAV vaccine at each of the vaccination visits (Visits 1, 3, and 7) according to Table A.

Subjects received vaccinations using a 0-, 2-, 6-month schedule. Group 1 received bivalent rLP2086 at Month 0 (Day 1) followed by subsequent vaccinations at Months 2 and 6. Group 2 received HAV vaccine at Month 0 and Month 6 and an injection with saline at Month 2 to maintain the study blind. Age-specific doses of HAV vaccine were supplied according to country-specific guidelines.

The primary endpoints for the safety population were the percentage of subjects with at least 1 serious adverse event (SAE) that occurred during the time period from the first study vaccination through 6 months after the last study vaccination and the percentage of subjects with at least 1 medically attended adverse event (AE) that occurred during the time period within 30 days after each vaccination.

Overall, the proportion of subjects reporting at least 1 SAE within 30 days of each vaccination was ≤3.0% for both groups and similar between bivalent rLP2086 and HAV vaccine/saline control. Throughout the study, a numerically greater proportion of SAEs were reported by subjects in Group 2 (HAV vaccine/saline) than Group 1 (bivalent rLP2086) (2.52% and 1.55%, respectively). SAEs were also reported by a numerically larger proportion of subjects in Group 2 compared to Group 1 during the vaccination and follow-up phases. Overall, the percentage of AEs reported during the vaccination phase was higher in Group 1 compared to Group 2. Subjects in Group 1 reported more reactogenicity AEs than subjects in Group 2 during the vaccination phase. Reactogenicity AEs were defined as any AE with an onset within 7 days after vaccination and matched a predefined preferred term list. No new safety concerns were identified in this study for the group that received bivalent rLP2086. The data from this study demonstrate that bivalent rLP2086 is safe and well tolerated in subjects aged ≥10 to <26 years.

Example 13: Immunogenicity of Meningococcal Serogroup B Bivalent rLP2086 Vaccine in Healthy Adolescents Given Concomitantly with Other Licensed Vaccines

Background: Bivalent rLP2086 (TRUMENBA®) a N. meningitidis serogroup B (MnB) factor H binding protein (fHBP) vaccine, is approved in the US for prevention of invasive MnB disease in 10-25 years olds. Concomitant administration of recommended vaccines in this population improves compliance and maximizes public health impact.

Objective: To demonstrate noninferiority of immune responses (based on geometric mean [GM] titers) induced by GARDASIL® (HPV4), or MENACTRA® (meningococcal Groups A, C, Y and W-135, MCV4) and ADACEL® (Tdap), when given concomitantly with TRUMENBA.

Design/Methods: Study 1: subjects (11-<18 years) received TRUMENBA+HPV4, TRUMENBA, or HPV4 at months 0, 2 and 6. Subjects receiving only TRUMENBA or HPV4 received saline injections to maintain blinding. Study 2: subjects (10-<13 years) received MCV4+Tdap+TRUMENBA, MCV4+Tdap, or TRUMENBA. TRUMENBA was administered at months 0, 2 and 6. MCV4 and Tdap were given at month 0. Groups receiving only MCV4 and Tdap, or TRUMENBA received saline injections at months 0, 2, and 6 to maintain blinding. Immune responses were assessed after final vaccine administration. In both studies, noninferiority was achieved if the lower bound of the 95% CI of the GM ratio between groups was >0.67 (a 1.5-fold noninferiority margin).

Results: The noninferiority criterion was met for all HPV antigens except HPV18 (lower 95% Cl=0.62) and for all Tdap and MCV4 antigens when these vaccines were coadministered with TRUMENBA. Seroconversion rates for all HPV antigens were 99% when HPV4 was given alone or with TRUMENBA. The noninferiority criterion was met for MnB hSBA responses when TRUMENBA was coadministered with licensed vaccines (Table).

Data support coadministration of TRUMENBA with HPV4, MCV4, and Tdap vaccines recommended for US adolescents.

TABLE Comparison of Geometric Means at 1 Month After Last Vaccination N^(a) GM^(b) N^(a) GM^(b) N^(a) GM^(b) Ratio^(c) (95% CI)^(d) Study 1 Bivalent Bivalent rLP2086 + rLP2086 + Saline + HPV4 Saline HPV4 (Group 1) (Group 2) (Group 3) HPV Antigens^(e) (Group 1 vs Group 3) HPV-6 813 451.8 NA NA 423 550.3 0.82 (0.72, 0.94) HPV-11 813 892.9 NA NA 423 1084.3 0.82 (0.74, 0.91) HPV-16 813 3695.4 NA NA 423 4763.4 0.78 (0.68, 0.88) HPV-18 813 744.0 NA NA 423 1047.4 0.71 (0.62, 0.81) hSBA MnB Strains [Variants]^(f) (Group 1 vs Group 2) PMB80 [A22] 803 53.3 801 57.8 NA NA 0.92 (0.85, 1.00) PMB2948 [B24] 788 25.8 793 28.0 NA NA 0.92 (0.84, 1.01) Study 2 Saline + MCV4 + Tdap + Saline + Bivalent MCV4 + Tdap + Bivalent rLP2086 Saline rLP2086 (Group 1) (Group 2) (Group 3) Tdap Antigens^(g) (Group 1 vs Group 2) Diphtheria toxoid 778 9.3 780 9.8 NA NA 0.94 (0.86, 1.03) Tetanus toxoid 778 9.4 780 10.3 NA NA 0.92 (0.85, 0.99) Pertussis toxoid 778 13.2 780 14.2 NA NA 0.93 (0.85, 1.02) Pertussis filamentous 778 112.0 780 122.9 NA NA 0.91 (0.84, hemagglutinin 0.98) Pertussis pertactin 778 202.0 780 228.9 NA NA 0.88 (0.80, 0.98) Pertussis fimbriae 778 138.1 780 154.2 NA NA 0.90 (0.74, agglutinogens types 2 + 3 1.08) rSBA MCV4 Antigens^(h) (Group 1 vs Group 2) Serogroup A 763 4647.3 772 5113.0 NA NA 0.91 (0.82, 1.01) Serogroup C 768 1679.2 767 1650.2 NA NA 1.02 (0.90, 1.15) Serogroup Y 771 2212.6 770 2244.9 NA NA 0.99 (0.89, 1.09) Serogroup W-135 751 5925.1 765 6367.9 NA NA 0.93 (0.83, 1.04) hSBA MnB Strains^(f) [Variant] (Group 1 vs Group 3) PMB80 [A22] 679 45.9 NA NA 674 49.7 0.92 (0.84, 1.02) PMB2948 [B24] 670 24.8 NA NA 656 27.4 0.90 (0.82, 1.00) CI = confidence interval; GMT = geometric mean titer; HPV = human papillomavirus, HPV4 = human papillomavirus quadrivalent vaccine; MCV4 = meningococcal (Groups A, C, Y and W-135) polysaccharide diphtheria toxoid conjugate vaccine; MnB = Neisseria meningitidis serogroup B; NA = not applicable; Tdap = tetanus toxoid, reduced diphtheria toxoid and acellular pertussis vaccine adsorbed. ^(a)N = number of subjects with valid and determinate assay results for the given antigen or strain. ^(b)Geometric means were calculated using all subjects with valid and determinate assay results at 1 month after Vaccination 3. ^(c)Ratios of GMs. ^(d)Confidence Intervals (CIs) for the ratio are back transformations of a confidence interval based on the Student t distribution for the mean difference of the logarithms of the measures (Group 1-Group 3 for HPV titers; Group 1-Group 2 for Tdap/MCV4 antigens). ^(e)Immune responses to HPV antigens assessed in a competitive LUMINEX immunoassay 1 month after the third Gardasil ® vaccination. ^(f)Immune responses to MnB antigens assessed in a serum bactericidal assay using human complement 1 month after the third bivalent rLP2086 vaccination. ^(g)Immune responses to diphtheria, tetanus, and pertussis antigens assessed in a LUMINEX assay 1 month after the single ADACEL ® vaccination. ^(h)Immune responses to MCV4 antigens assessed in a serum bactericidal assay using rabbit complement 1 month after the single MENACTRA ® vaccination.

Example 14: Phase 3 Trial of Immunogenicity of Bivalent rLP2086, A Meningococcal Serogroup B Vaccine, in Adolescents: Bactericidal Activity Against a Panel of Antigenically Diverse Strains

Background:

Bivalent rLP2086, which targets factor H binding proteins (fHBP), is approved in the US to prevent meningococcal serogroup B (MnB) disease in 10-25-year-olds. Broad protection with bivalent rLP2086 was initially demonstrated in hSBAs with 4 diverse invasive MnB strains expressing fHBPs with sequences different from vaccine antigens. In this pivotal phase 3 trial, broad coverage against MnB disease is further supported by hSBA data with 10 additional MnB test strains representing the diversity of circulating invasive MnB strains (ClinicalTrials.gov: NCT01830855).

Methods:

Healthy subjects aged 10-<19 years were randomized to receive bivalent rLP2086 at 0, 2, and 6 months, or hepatitis A virus vaccine at 0 and 6 months and saline at 2 months. Immune responses were assessed in hSBAs with 4 primary MnB test strains (primary endpoint; N=1210-1266) and in a population subset using 10 secondary MnB test strains (secondary endpoint; N=266-281). All strains expressed vaccine-heterologous fHBP.

Results:

hSBA responses 1 month after doses 2 and 3 among bivalent rLP2086 recipients against 4 primary MnB test strains measured by hSBA titers ≥LLOQ were 64.0%-99.1% and 87.1%-99.5%, respectively (Table). hSBA responses to 10 secondary MnB test strains were 61.1%-100.0% and 75.1%-98.6% 1 month after dose 2 and 3, respectively. hSBA GMTs for each secondary test strain increased from 4.5-11.4 at baseline to 22.1-93.5 after dose 3.

TABLE Immunogenicity of bivalent rLP2086 against secondary MnB test strains Strain hSBA titers ≥LLOQ, % (1:8 or 1:16) hSBA GMTs (fHBP Variant) N Baseline After dose 2* After dose 3 Baseline After dose 3 Primary strains PMB80 (A22) 1266 33.2 94.3 97.8 12.6 86.8 PMB2001 1229 27.5 99.1 99.5 8.4 222.5 (A56) PMB2948 1250 6.4 66.4 87.1 4.5 24.1 (B24) PMB2707 1210 3.6 64.0 89.3 4.3 50.9 (B44) Secondary strains PMB3175 278 17.5 100.0 98.6 5.7 93.5 (A29) PMB3010 280 9.4 84.0 95.7 9.3 78.6 (A06) PMB3040 280 43.1 93.8 96.4 11.4 63.5 (A07) PMB824 (A12) 277 3.9 67.4 75.1 8.4 22.3 PMB1672 266 20.7 65.6 87.2 5.9 31.0 (A15) PMB1989 275 11.3 84.5 92.7 9.1 57.6 (A19) PMB1256 279 4.3 61.1 92.5 4.5 51.7 (B03) PMB866 (B09) 276 15.2 76.3 86.2 5.2 22.9 PMB431 (B15) 281 28.7 96.8 98.2 7.3 47.7 PMB648 (B16) 278 7.6 61.6 81.7 4.7 22.1 *For the secondary strains, subset of subjects; n = 86-97. LLOQ = 1:16 for A06, A12, A19, and A22; 1:8 for A07, A15, A29, A56, B03, B09, B15, B16, B24, and B44.

Conclusions:

Bivalent rLP2086 vaccination resulted in robust immune responses against diverse MnB strains heterologous to vaccine antigens after 2 and 3 doses. A high proportion of individuals developed protective hSBA titres greater than the correlate of protection (hSBA titre ≥4) to 10 additional MnB test strains. Collectively, these phase 3 immunogenicity data support the broad protection afforded by bivalent rLP2086 against MnB disease in adolescents.

Example 15: Phase 3 Trial of Immunogenicity of Bivalent rLP2086, A Meningococcal Serogroup B Vaccine, in Young Adults: Bactericidal Activity Against a Panel of Antigenically Diverse Strains

Background:

Bivalent rLP2086, which targets factor H binding proteins (fHBP), is approved in the US to prevent meningococcal serogroup B (MnB) disease in 10-25-year-olds. Broad protection with bivalent rLP2086 was initially demonstrated in hSBAs with 4 diverse invasive MnB strains expressing fHBPs with sequences different from vaccine antigens. In this pivotal phase 3 trial, broad coverage against MnB disease is further supported by hSBA data with 10 additional MnB test strains representing the diversity of circulating invasive MnB strains (ClinicalTrials.gov: NCT01352845).

Methods:

Healthy subjects aged 18-<26 years were randomized to receive bivalent rLP2086 or saline at 0, 2, and 6 months. Immune responses were assessed in hSBAs with 4 primary MnB test strains (primary endpoint; N=1702-1714) and in a population subset (N=273-284) using 10 secondary MnB test strains (secondary endpoint; N=273-284). All strains expressed vaccine-heterologous fHBP.

Results:

hSBA responses 1 month after dose 2 and 3 among bivalent rLP2086 recipients against 4 primary MnB test strains as measured by hSBA titers ≥LLOQ were 68.3%-97.4% and 87.4%-99.4%, respectively (Table). hSBA responses to 10 secondary MnB test strains were 51.6%-97.9% and 71.3%-99.3% 1 month after dose 2 and 3, respectively. hSBA GMTs for each secondary strain increased from 5.1-13.9 at baseline to 20.6-96.3 after dose 3.

TABLE Immunogenicity of bivalent rLP2086 against secondary MnB test strains Strain hSBA titers ≥LLOQ, % (1:8 or 1:16) hSBA GMTs (fHBP Variant) N Baseline After dose 2* After dose 3 Baseline After dose 3 Primary strains PMB80 (A22) 1714 33.6 84.7 93.5 12.8 74.3 PMB2001 (A56) 1709 32.2 97.4 99.4 8.8 176.7 PMB2948 (B24) 1702 33.1 86.5 95.1 7.6 49.5 PMB2707 (B44) 1703 11.0 68.3 87.4 4.8 47.6 Secondary strains PMB3175 (A29) 283 31.1 96.8 99.3 7.1 96.3 PMB3010 (A06) 275 16.0 77.8 92.0 10.3 69.9 PMB3040 (A07) 277 55.8 97.9 95.7 13.9 60.4 PMB824 (A12) 275 5.0 57.6 71.3 8.4 20.6 PMB1672 (A15) 279 37.3 83.2 91.8 8.0 43.1 PMB1989 (A19) 284 28.8 87.4 95.8 12.1 87.3 PMB1256 (B03) 273 11.2 57.9 86.4 5.1 49.8 PMB866 (B09) 274 23.5 65.3 77.0 6.1 23.3 PMB431 (B15) 276 43.8 86.5 96.7 9.1 49.4 PMB648 (B16) 273 21.9 51.6 78.0 6.2 26.5 *For the secondary strains, subset of subjects; n = 90-96. LLOQ = 1:16 for A06, A12, A19, and A22; 1:8 for A07, A15, A29, A56, B03, B09, B15, B16, B24, and B44.

Conclusions:

Bivalent rLP2086 vaccination elicited robust immune responses against diverse MnB strains expressing fHBP variants heterologous to vaccine antigens after 2 and 3 doses. A high proportion of individuals developed protective hSBA titres greater than the correlate of protection (hSBA titre ≥4) to 10 additional MnB test strains. Collectively, these phase 3 immunogenicity data support the broad protection afforded by bivalent rLP2086 against MnB disease in young adults. 

What is claimed is:
 1. A method of inducing bactericidal immune response against a Neisseria meningitidis serogroup B subfamily A strain and against a Neisseria meningitidis serogroup B subfamily B strain in a human comprising concomitantly administering an effective amount of a N. meningitidis rLP2086 composition and three additional immunogenic compositions, wherein said N. meningitidis rLP2086 composition comprises (a) a first lipidated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 and (b) a second lipidated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, wherein the N. meningitidis rLP2086 composition comprises about 2.8 molar ratio of polysorbate-80, wherein the three additional immunogenic compositions comprise (i) an immunogenic composition comprising four distinct and separately made carrier protein-capsular polysaccharide conjugates against Neisseria meningitidis serogroup A, Neisseria meningitidis serogroup C, Neisseria meningitidis serogroup Y, and Neisseria meningitidis serogroup W135; (ii) an immunogenic composition against diphtheria, tetanus, and pertussis comprising diphtheria toxoid, tetanus toxoid, and acellular pertussis antigens; and (iii) an immunogenic composition against human papillomavirus (HPV).
 2. The method of claim 1, wherein the four carrier protein-capsular polysaccharide conjugates are a first conjugate comprising serogroup A Neisseria meningitidis capsular polysaccharide conjugated a carrier protein, a second conjugate comprising serogroup C Neisseria meningitidis capsular polysaccharide conjugated a carrier protein, a third conjugate comprising serogroup Y Neisseria meningitidis capsular polysaccharide conjugated a carrier protein, and a fourth conjugate comprising serogroup W135 Neisseria meningitidis capsular polysaccharide conjugated a carrier protein, wherein the carrier protein is CRM197 or diphtheria toxoid.
 3. The method of claim 1, wherein the four carrier protein-capsular polysaccharide conjugates are a first conjugate comprising serogroup A Neisseria meningitidis capsular polysaccharide conjugated a carrier protein, a second conjugate comprising serogroup C Neisseria meningitidis capsular polysaccharide conjugated a carrier protein, a third conjugate comprising serogroup Y Neisseria meningitidis capsular polysaccharide conjugated a carrier protein, and a fourth conjugate comprising serogroup W135 Neisseria meningitidis capsular polysaccharide conjugated a carrier protein, wherein the carrier protein is tetanus toxoid.
 4. The method of claim 2 or claim 3, wherein the additional immunogenic composition comprising the mixture of the four conjugates is a liquid composition.
 5. The method of claim 2 or claim 3, wherein the additional immunogenic composition comprising the mixture of the four conjugates is not lyophilized.
 6. The method of claim 1, wherein the immunogenic composition against human papillomavirus is GARDASIL which generates an immune response against at least one of human papillomavirus types 6, 11, 16 and 18 or any combination thereof.
 7. The method of claim 1, wherein the immunogenic composition against diphtheria, tetanus, and pertussis is Tdap vaccine.
 8. The method of claim 2 or claim 3, wherein the method induces an immune response against at least one strain of serogroup A Neisseria meningitidis, at least one strain of serogroup C Neisseria meningitidis, at least one strain of serogroup Y Neisseria meningitidis, at least one strain of serogroup W135 Neisseria meningitidis, or any combination thereof.
 9. The method of claim 1, wherein the immunogenic composition (ii) further comprises inactivated poliomyelitis virus.
 10. The method of claim 9, wherein the method induces an immune response against at least one of diphtheria, tetanus, pertussis, poliomyelitis, or any combination thereof in the human.
 11. The method according to claim 1, wherein the N. meningitidis rLP2086 composition further comprises aluminum.
 12. The method according to claim 1, wherein the N. meningitidis rLP2086 composition further comprises histidine buffer.
 13. The method according to claim 1, wherein the N. meningitidis rLP2086 composition further comprises sodium chloride.
 14. The method according to claim 1, wherein the N. meningitidis rLP2086 composition comprises about 120 μg/ml of the first polypeptide; about 120 μg/ml of the second polypeptide; about 2.8 molar ratio of polysorbate-80; about 0.5 mg/ml aluminum; about 10 mM histidine; and about 150 mM sodium chloride.
 15. The method according to claim 1, wherein the N. meningitidis rLP2086 composition comprises about 60 μg/ml of the first polypeptide; about 60 μg/ml of the second polypeptide; about 18 μg of polysorbate-80; about 250 μg/mIaluminum; about 780 μg histidine; and about 4380 μg sodium chloride.
 16. The method according to claim 1, wherein the N. meningitidis rLP2086 composition induces a bactericidal immune response against at least one of N. meningitidis serogroup B A22, A56, B24, B44 strains, or any combination thereof.
 17. The method according to claim 1, wherein the N. meningitidis rLP2086 composition induces a bactericidal immune response against N. meningitidis serogroup B A22.
 18. The method according to claim 1, wherein the N. meningitidis rLP2086 composition induces a bactericidal immune response against N. meningitidis serogroup B B24.
 19. The method according to claim 1, wherein the N. meningitidis rLP2086 composition induces a bactericidal immune response against at least one of N. meningitidis serogroup B B24, B16, B44, A22, B03, B09, A12, A19, A05, A07, B153 strains, or any combination thereof.
 20. The method according to claim 1, wherein the effective amount of the N. meningitidis rLP2086 composition comprises one dose.
 21. The method according to claim 1, wherein the effective amount of the N. meningitidis rLP2086 composition comprises two doses.
 22. The method according to claim 21, wherein the effective amount of the N. meningitidis rLP2086 composition further comprises a booster dose.
 23. The method according to claim 1, wherein the effective amount of the N. meningitidis rLP2086 composition comprises at most three doses.
 24. The method according to claim 1, wherein the N. meningitidis rLP2086 composition is administered within 24 hours of administration of at least one of the three additional immunogenic compositions.
 25. The method according to claim 1, wherein the N. meningitidis rLP2086 composition is simultaneously administered with at least one of the three additional immunogenic compositions.
 26. The method according to claim 1, wherein a first dose of the N. meningitidis rLP2086 composition is concomitantly administered with at least one of the three additional immunogenic compositions.
 27. The method according to claim 26, wherein the first dose of the N. meningitidis rLP2086 composition is simultaneously administered with at least one of the three additional immunogenic compositions. 